Methods and composition for secretion of heterologous polypeptides

ABSTRACT

The present invention relates generally to the fields of molecular biology and protein technology. More specifically, the invention concerns signal sequences for the secretion of heterologous polypeptide from bacteria. The invention also concerns recombinant polypeptides and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application to U.S. patent applicationSer. No. 12/940,825, filed on Nov. 5, 2010, which claims priority toU.S. patent application No. 61/258,565, filed on Nov. 5, 2009, thecontents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2012, isnamed P4386R1D1_SequenceListing.TXT and is 33,149 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology and protein technology. More specifically, the inventionconcerns signal sequences for the secretion of heterologous polypeptidesfrom bacteria. The invention also concerns prokaryotically producedrecombinant polypeptides and uses thereof.

BACKGROUND OF THE INVENTION

Secretion of heterologous polypeptides into the periplasmic space of E.coli and other prokaryotes or into their culture media is subject to avariety of parameters. Typically, vectors for secretion of a polypeptideof interest are engineered to position DNA encoding a secretory signalsequence 5′ to the DNA encoding the polypeptide of interest.

Recent years have seen increasing promises of using heterologouspolypeptide, for example, antibodies, as diagnostic and therapeuticagents for various disorders and diseases. Many research and clinicalapplications require large quantities of functional polypeptide, thuscalling for scaled-up, yet economic systems for polypeptide production.Particularly useful is the recombinant production of antibodies using avariety of expression hosts, ranging from prokaryotes such as E. coli orB. subtilis, to yeast, plants, insect cells and mammalian cells.Kipriyanov and Little (1999) Mol. Biotech. 12:173-201.

Compared to other polypeptide production systems, bacteria, particularlyE. coli, provides many unique advantages. The raw materials used (i.e.bacterial cells) are inexpensive and easy to grow, therefore reducingthe cost of products. Prokaryotic hosts grow much faster than, e.g.,mammalian cells, allowing quicker analysis of genetic manipulations.Shorter generation time and ease of scaling up also make bacterialfermentation a more attractive means for large quantity proteinproduction. The genomic structure and biological activity of manybacterial species including E. coli have been well-studied and a widerange of suitable vectors are available, making expression of adesirable antibody more convenient. Compared with eukaryotes, fewersteps are involved in the production process, including the manipulationof recombinant genes, stable transformation of multiple copies into thehost, expression induction and characterization of the products.Pluckthun and Pack (1997) Immunotech 3:83-105.

Various approaches have been used to make recombinant polypeptides inbacteria. Recombinant proteins can be obtained from bacteria eitherthrough refolding of inclusion bodies expressed in the cytoplasm, orthrough expression followed by secretion to the bacterial periplasm. Thechoice between secretion and refolding is generally guided by severalconsiderations. Secretion is usually the faster and more commonly usedstrategy for producing antibodies. Kipriyanov and Little (1999), supra.

Antibody expression in prokaryotic systems can be carried out indifferent scales. The shake-flask cultures (in the 2-5 liter-range)typically generate less than 5 mg/liter products. Carter et al. (1992)Bio/Technology 10:12-16 developed a high cell-density fermentationsystem in which high-level expression (up to 2 g/liter) of antibodyfragments was obtained. The gram per liter titers of Fab′ obtained byCarter et al. is due largely to higher cell densities resulting from themore precisely controlled environment of a fermentor than that of asimple shake flask. The system contains a dicistronic operon designed toco-express the light chain and heavy chain fragments. The dicistronicoperon is under the control of a single E. coli phoA promoter which isinducible by phosphate starvation. Each antibody chain is preceded bythe E. coli heat-stable enterotoxin II (stII) signal sequence to directsecretion to the periplasmic space.

For general reviews of antibody production in E. coli, see Pluckthun andPack (1997) Immunotech 3:83-105; Pluckthun et al. (1996) in ANTIBODYENGINEERING: A PRACTICAL APPROACH, pp 203-252 (Oxford Press); Pluckthun(1994) in HANDBOOK OF EXP PHARMCOL VOL 3: THE PHARMCOL OF MONOCLONALANTIBODIES, pp 269-315 (ed. M. Rosenberg and G.P. Moore;Springer-Verlag, Berlin).

Many biological assays (such as X-ray crystallography) and clinicalapplications (such as protein therapy) require large amounts of protein.Accordingly, a need exists for high yield yet simple systems forproducing properly assembled, soluble and functional heterologouspolypeptides, such as antibodies.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides a novel means for increasing production ofheterologous proteins comprising use of novel translational initiationregion (TIR) variants, including TIR variants comprisingco-translational secretion signal peptides (signal peptides that directtranslocation in a co-translational manner) and/or TIR variantscomprising post-translational secretion signal peptides (signal peptidesthat direct translocation in a post-translational manner). In addition,demonstrated herein is increased antibody production using vectorscomprising antibody light chain operably linked to a TIR comprising aco- or post-translational secretion signal peptide and an antibody heavychain operably linked to a TIR comprising a co-translational secretionsignal peptide for peak expression. Novel TIR variants are also providedherein.

In one aspect, the invention provides variant translation initiationregions. In some embodiments, the variant comprises a varianttranslation initiation region (in some embodiments, a prokaryoticpost-translational secretion signal sequence or a prokaryoticco-translational secretion signal sequence). In some embodiments, thevariant comprises nucleic acid variants of a secretion signal sequence,such as PhoA, MalE, DsbA or STII. In some embodiments, the variantfurther comprises a MlaI, BssHII, or XbaI restriction site. In someembodiments, the variant comprises a translation initiation regionvariant comprising a sequence shown Table 3.

In one aspect, the invention provides variant secretion signalsequences. In some embodiments, the secretion signal sequence is aprokaryotic post-translational secretion signal sequence or aprokaryotic co-translational secretion signal sequence. In someembodiments, the secretion signal sequence is a eukaryoticpost-translational secretion signal sequence or a eukaryoticco-translational secretion signal sequence. In some embodiments, thevariants are nucleic acid variants of a PhoA, MalE, DsbA or STIIsecretion signal sequence. In some embodiments, the variants comprise asecretion signal sequence shown in Table 3. The variant secretion signalsequences of the invention are suitable for use, for example, in any ofthe methods disclosed herein.

In another aspect, the invention provides a polynucleotide comprising atranslation initiation region of the invention. In some embodiments, thetranslation initiation region comprises sequence shown in Table 3 (e.g.,one of SEQ ID NOs 1-42). In some embodiments, the translation initiationregion comprises one of SEQ ID NOs. 1-14, 16-24, 26-39, 41-42. Thepolynucleotides are suitable for use, for example, in any of the methodsdisclosed herein.

In another aspect, the invention provides a polynucleotide comprising asecretion signal sequence of the invention. In some embodiments, thesecretion signal sequence comprises sequence shown in Table 3. (e.g.,one of SEQ ID NOs 1-42). In some embodiments, the translation initiationregion comprises one of SEQ ID NOs. 1-14, 16-24, 26-39, 41-42. Thepolynucleotides are suitable for use, for example, in any of the methodsdisclosed herein.

In another aspect, the invention provides a polynucleotide comprising atranslation initiation region of the invention operably linked to apolynucleotide encoding a heterologous polypeptide, whereby uponexpression of the heterologous polypeptide in a host cell (e.g., aprokaryotic host cell, e.g., an E. coli host cell), the heterologouspolypeptide is folded and assembled to form a biologically activeheterologous polypeptide. Examples of heterologous polypeptides arefurther disclosed herein. In some embodiments, the heterologouspolypeptide is an antibody heavy chain. In some embodiments, theheterologous polypeptide is an antibody light chain. In someembodiments, the heterologous polypeptide is an Fc polypeptide. In someembodiments, the heterologous polypeptide is a multimeric polypeptide.In some embodiments, the heterologous polypeptide is a heteromultimer.In some embodiments, the translation initiation region is anytranslation initiation region disclosed herein, e.g., a translationinitiation region comprising sequence shown in Table 3. In someembodiments, the translation initiation region comprises sequence of oneof SEQ ID NOs 1-42. In some embodiments, the translation initiationregion comprises sequence of one of SEQ ID NOs 1-14, 36-39, 41-42. Insome embodiments, the translation initiation region comprises a variantSTII, DsbI, PhoA, or MalE signal sequence.

In another aspect, the invention provides a polynucleotide comprising(1) a first translation initiation region (TIR) operably linked to apolynucleotide encoding a first heterologous polypeptide, wherein theTIR comprises a co-translation prokaryotic secretion signal sequence;and (2) a second TIR operably linked to a polynucleotide encoding ansecond heterologous, wherein the second TIR comprises a co-translationor post-translation prokaryotic secretion signal sequence, whereby uponexpression of the antibody in a host cell, the first and secondheterologous polypeptides are folded and assembled to form abiologically active polypeptide complex.

In another aspect, the invention provides a polynucleotide encoding anantibody, said polynucleotide comprising (1) a first translationinitiation region of the invention operably linked to a polynucleotideencoding an antibody heavy chain and (2) a second translation initiationregion operably linked to a polynucleotide encoding an antibody lightchain, whereby upon expression of the antibody in a host cell (e.g., aprokaryotic host cell, e.g., an E. coli host cell), the heavy and lightchains are folded and assembled to form a biologically active antibody.

In some embodiments, the first translation initiation region comprises aco-translational prokaryotic secretion signal sequence (e.g., a signalsequence that directs translation through the signal recognitionpeptide). In some embodiments, the first translation initiation regioncomprises a STII or DsbA signal sequence. In some embodiments, the firsttranslation initiation region comprises a DsbA signal sequence. In someembodiments, the first translation initiation region comprises a PhoA orMalE signal sequence. In some embodiments, the first translationinitiation region comprises sequence of one of SEQ ID NOs: 1-10 and36-42. In some embodiments, the first translation initiation regioncomprises sequence of one of SEQ ID NOs: 1-10 and 36-29 and 41 and 42.In some embodiments, the first translation initiation region comprisessequence of one of SEQ ID Nos 1-42. In some embodiments, the firsttranslation initiation region comprises sequence of one of SEQ ID Nos.1-14, 16-24, 26-39, 41-42.

In some embodiments, the second translation initiation region comprises(i) a co-translational prokaryotic secretion signal sequence or apost-translation prokaryotic secretion signal sequence (e.g., a signalsequence that directs translation through the sec pathway). In someembodiments, the second translation initiation region comprises a STII,DsbA, MalE or PhoA signal sequence. In some embodiments, the secondtranslation initiation region comprises a PhoA or MalE signal sequence.In some embodiments, the second translation initiation region comprisessequence of one of SEQ ID NOs 1-42. In some embodiments, the secondtranslation initiation region comprises sequence of one of SEQ ID NOs1-14, 16-24, 26-39, 41-42.

In some embodiments, the polynucleotide encoding an antibody furthercomprises (3) a third translation initiation region operably linked to apolynucleotide encoding a Fc polypeptide. In some embodiments, the thirdtranslation initiation region comprises a STII, PhoA or DsbA signalsequence. In some embodiments, the third translation initiation regioncomprises a DsbA signal sequence. In some embodiments, the thirdtranslation initiation region comprises a PhoA signal sequence.

In another aspect, the invention provides polynucleotide comprising (1)a first translation initiation region (TIR) operably linked to apolynucleotide encoding an antibody heavy chain, wherein the TIRcomprises a co-translation prokaryotic secretion signal sequence; and(2) a second TIR operably linked to a polynucleotide encoding anantibody light chain, wherein the second TIR comprises a co-translationor post-translation prokaryotic secretion signal sequence, whereby uponexpression of the antibody in a host cell, the heavy and light chainsare folded and assembled to form a biologically active antibody.

In another aspect, the invention provides a polynucleotide encoding anantibody fragment (such as a monovalent antibody fragment), saidpolynucleotide comprising (1) a first translation initiation region ofthe invention operably linked to a polynucleotide encoding an antibodyheavy chain; (2) a second translation initiation region operably linkedto a polynucleotide encoding an antibody light chain; and (3) a thirdtranslation initiation region operably linked to a polynucleotideencoding a Fc polypeptide, whereby upon expression of the antibody in ahost cell (e.g., a prokaryotic host cell), the heavy chain, light chainand Fc polypeptide are folded and assembled to form a biologicallyactive antibody (such as an one-armed antibody). In some embodiments,the third translation initiation region comprises a co-translationalprokaryotic secretion signal sequence or a post-translationalprokaryotic secretion signal sequence. In some embodiments, the thirdtranslation initiation region comprises a STII, PhoA, MalE, or DsbAsignal sequence. In some embodiments, the third translation initiationregion comprises a DsbA signal sequence. In some embodiments, the thirdtranslation initiation region comprises a PhoA signal sequence. In someembodiments, the third translation initiation region comprises sequenceof one of SEQ ID Nos 1-42. In some embodiments, the third translationinitiation region comprises sequence of one of SEQ ID Nos. 1-14, 16-24,26-39, 41-42.

In another aspect, the invention provides a polynucleotide encoding anantibody, said polynucleotide comprising (1) a first translationinitiation region of the invention operably linked to a polynucleotideencoding an antibody heavy chain, wherein the first translationinitiation region comprises a STII or DsbA signal sequence and (2) asecond translation initiation region operably linked to a polynucleotideencoding an antibody light chain, wherein the second translationinitiation region comprises a STII, DsbA, MalE or PhoA signal sequence,whereby upon expression of the antibody in a host cell (e.g., aprokaryotic host cell), the light and heavy chains are folded andassembled to form a biologically active antibody. In some embodiments,the first translation initiation region comprises a DsbA signal sequenceand the second translation initiation region comprises a MalE or PhoAsignal sequence. In some embodiments, the polynucleotide encoding anantibody further comprises (3) a third translation initiation regionoperably linked to a polynucleotide encoding a Fc polypeptide. In someembodiments, the third translation initiation region comprises a STII,PhoA or DsbA signal sequence. In some embodiments, the third translationinitiation region comprises a PhoA signal sequence. In some embodiments,the third translation initiation region comprises a DsbA signalsequence.

In some embodiments, the translational strength of said varianttranslation initiation region is less than the translational strength ofthe wild-type translation initiation region. In some embodiments, thetranslational strength of said variant translation initiation region isgreater than the translational strength of the wild-type translationinitiation region. In some embodiments, the amino acid sequence of thetranslation initiation variant is not altered relative to wild-typeamino acid sequence. In some embodiments, the amino acid sequence of thetranslation initiation variant is altered relative to wild-type aminoacid sequence. In some embodiments, the translation initiation regionincludes a prokaryotic secretion signal sequence. In some embodiments,the first and second translational initiation regions (and in someembodiment, the third translational initiation region) provideapproximately equal translational strengths. In some embodiments, therelative translation strength is about one or two. In some embodimentsthe relative translation strength is about one. In some embodiments, therelative translation strength is about two. In some embodiments, therelative translation strength is one and/or two. In some embodiments,the relative translation strength is about three or about four. In someembodiments, the relative translation strength is selected from one ormore of one, two, three, four, five, or more (such as six or seven ormore).

In some embodiments, the polynucleotide of the invention furthercomprises a promoter operably linked to the heterologous polypeptide. Insome embodiments, the promoter is a prokaryotic promoter selected fromthe group consisting of phoA, tac, lpp, lac-lpp, lac, ara, trp, and T7promoter. In some embodiments, the promoter is a phoA promoter. In someembodiments involving expression of antibody heavy and light chain, thepolynucleotide further comprises (a) a first promoter, wherein the firstpromoter is operably linked to a light chain and (b) a second promoter,wherein the second promoter is operably linked to a heavy chain. In someembodiments, the first and second promoters are both phoA promoters. Insome embodiments involving expression of antibody heavy and light chainand Fc polypeptide, the polynucleotide further comprises (c) a thirdpromoter, wherein the third promoter is operably linked to a Fcpolypeptide. In some embodiments, the third promoter is a Fcpolypeptide.

When expressing polypeptides that comprise more than one polypeptide(e.g., an antibody comprising a heavy chain and light chain), thepolynucleotide for expressing the polypeptide may be a polycistronicpolynucleotide (ie, a single polynucleotide that contains and expressesmultiple cistrons under the regulatory control of a single promoter). Acommon example of a polycistronic vector is a “dicistronic” vector thatcontains and expresses two different polypeptides under the control ofone promoter. Upon expression of a dicistronic or poycistronic vector,multiple coding regions (eg, genes) are first transcribed as a singletranscriptional unit, and then translated separately. A cistron refersto a genetic element broadly equivalent to a translation unit comprisingthe nucleotide sequence coding for a polypeptide chain and adjacentcontrol regions (including, e.g., a TIR). In other embodiments, thepolynucleotide may comprise separate cistrons, which refers to a singlepolynucleotide comprising at least two separate promoter-citron pairs,wherein each cistron is under the control of its own promoter. Uponexpression of a separate cistron expression vector, both transcriptionand translation processes of different genes are separate andindependent. In yet another embodiments, the polynucleotide may comprisea polycistronic portion and a separate cistron portion.

In yet another aspect, the invention provides vectors comprisingpolynucleotide of the invention. In some embodiments, the vectors areexpression vectors.

In a further aspect, the invention provides compositions comprising oneor more polynucleotides of the invention and a carrier. In oneembodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprisingpolynucleotide or vector of the invention. In some embodiments, the hostcells comprise polynucleotide of the invention encoding an antibody (insome embodiments, a bispecific or one-armed antibody). The host cell maycomprise one or more polynucleotides collectively encoding the antibody.A vector can be of any type, for example, a recombinant vector such asan expression vector. Any of a variety of host cells can be used. In oneembodiment, a host cell is a prokaryotic cell, for example, E. coli. Insome embodiments, the E. coli is of a strain deficient in endogenousprotease activities. In some embodiments, the genotype of the E. colilacks degP and prc genes and harbors a mutant spr gene.

In some embodiments, the host cell further comprises a polynucleotideencoding a prokaryotic chaperone protein (such as Dsb proteins (DsbA,DsbB, DsbC, DsbD, FkpA and/or DsbG). In some embodiments, chaperonprotein is overexpressed in the host cell. In some embodiments, thechaperone protein is Dsb A and/or DsbC.

In one aspect, the host cell comprises one or more polynucleotidescollectively encoding a one-armed antibody. In one embodiment, a singlepolynucleotide encodes (a) the light and heavy chain components of theone armed antibody, and (b) the Fc polypeptide. In one embodiment, asingle polynucleotide encodes the light chain and Fc polypeptidecomponents of the one armed antibody, and a separate polynucleotideencodes the heavy chain polypeptide. In one embodiment, a singlepolynucleotide encodes the heavy chain and Fc polypeptide components ofthe one-armed antibody and a separate polynucleotide encodes the lightchain component of the one-armed antibody. In one embodiment, separatepolynucleotides encode the light chain component of the one-armedantibody, the heavy chain component of the one-armed antibody and the Fcpolypeptide, respectively.

Heterologous polypeptides are described herein. In some embodiments, theheterologous polypeptide is an antibody. In some embodiments, theantibody is a monoclonal antibody. In other embodiments, the antibody isa polyclonal antibody. In some embodiments, the antibody is selectedfrom the group consisting of a chimeric antibody, an affinity maturedantibody, a humanized antibody, and a human antibody. In certainembodiments, the antibody is a bispecific antibody. In certainembodiments, the antibody is an antibody fragment. In some embodiments,the antibody is a monovalent antibody. In some embodiments, the antibodyis a Fab, Fab′, Fab′-SH, F(ab′)₂, or scFv. In some embodiments, theantibody is a one-armed antibody (i.e., the heavy chain variable domainand the light chain variable domain form a single antigen binding arm)comprising an Fc region, wherein the Fc region comprises a first and asecond Fc polypeptide, wherein the first and second Fc polypeptides arepresent in a complex and form a Fc region that increases stability ofsaid antibody fragment compared to a Fab molecule comprising saidantigen binding arm.

In some embodiments, the antibody binds (in some embodiments,specifically binds) c-met. In some embodiments, the anti-c-met antibodycomprises (a) a first polypeptide comprising a heavy chain variabledomain having the sequence:EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS(SEQ ID NO:43), CH1 sequence, and a first Fc polypeptide; (b) a secondpolypeptide comprising a light chain variable domain having thesequence:DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR(SEQ ID NO:44), and CL1 sequence; and (c) a third polypeptide comprisinga second Fc polypeptide, wherein the heavy chain variable domain and thelight chain variable domain are present as a complex and form a singleantigen binding arm, wherein the first and second Fc polypeptides arepresent in a complex and form a Fc region that increases stability ofsaid antibody fragment compared to a Fab molecule comprising saidantigen binding arm. In some embodiments, the first polypeptidecomprises the Fc sequence depicted in FIG. 7 (SEQ ID NO: 68) and thesecond polypeptide comprises the Fc sequence depicted in FIG. 8 (SEQ IDNO: 47). In some embodiments, the first polypeptide comprises the Fcsequence depicted in FIG. 8 (SEQ ID NO: 47) and the second polypeptidecomprises the Fc sequence depicted in FIG. 7 (SEQ ID NO: 68).

In some embodiments, the anti-c-met antibody comprises (a) a firstpolypeptide comprising a heavy chain variable domain, said polypeptidecomprising the sequence:EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 45); (b) a second polypeptide comprising a light chainvariable domain, the polypeptide comprising the sequenceDIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:46); and a third polypeptide comprising a Fc polypeptide, thepolypeptide comprising the sequenceDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 47), wherein the heavy chain variable domain and the lightchain variable domain are present as a complex and form a single antigenbinding arm, wherein the first and second Fc polypeptides are present ina complex and form a Fc region that increases stability of said antibodyfragment compared to a Fab molecule comprising said antigen binding arm.

In one embodiment, the anti-c-met antibody comprises a heavy chainvariable domain comprising one or more of CDR1-HC, CDR2-HC and CDR3-HCsequence depicted in FIG. 7 (SEQ ID NO:52-53 & 66). In some embodiments,the antibody comprises a light chain variable domain comprising one ormore of CDR1-LC, CDR2-LC and CDR3-LC sequence depicted in FIG. 7 (SEQ IDNOs: 49-51). In some embodiments, the heavy chain variable domaincomprises FR1-HC, FR2-HC, FR3-HC and FR4-HC sequence depicted in FIG. 7(SEQ ID NOs: 62-65). In some embodiments, the light chain variabledomain comprises FR1-LC, FR2-LC, FR3-LC and FR4-LC sequence depicted inFIG. 7 (SEQ ID NO: 57-60).

In some embodiments, the antibody comprises at least one characteristicthat promotes heterodimerization, while minimizing homodimerization, ofthe Fc sequences within the antibody fragment. Such characteristic(s)improves yield and/or purity and/or homogeneity of the immunoglobulinpopulations obtainable by methods of the invention as described herein.In one embodiment, a first Fc polypeptide and a second Fc polypeptidemeet/interact at an interface. In some embodiments wherein the first andsecond Fc polypeptides meet at an interface, the interface of the secondFc polypeptide (sequence) comprises a protuberance (also termed a“knob”) which is positionable in a cavity (also termed a “hole”) in theinterface of the first Fc polypeptide (sequence). In one embodiment, theantibody comprises Fc mutations constituting “knobs” and “holes” asdescribed in WO2005/063816. For example, a hole mutation can be one ormore of T366A, L368A and/or Y407V in an Fc polypeptide, and a knobmutation can be T366W.

The invention also provides methods using the variant TIR and signalsequences of the invention. It is understood that any of the variantTIR, signal sequences and polynucleotides disclosed herein are suitablefor use in methods, e.g., methods of the invention disclosed herein. Ina further aspect, the invention provides methods of making aheterologous polypeptide of the invention. For example, the inventionprovides methods of making an a heterologous polypeptide (e.g., anantibody, which, as defined herein includes full length antibody andfragments thereof), said method comprising culturing a host cellcomprising a polynucleotide of the invention (e.g., a polynucleotidecomprising a translation initiation region) so that the polynucleotideis expressed, whereby upon expression of said polynucleotide in a hostcell (e.g. a prokaryotic host cell), the heterologous polypeptide isfolded to form a biologically active heterologous polypeptide. Inembodiments involving expression of antibodies, upon expression of saidpolynucleotide in a host cell, the light and heavy chains are folded andassembled to form a biologically active antibody. In some embodiments,the method further comprises recovering the heterologous polypeptide(e.g., an antibody) from the host cell culture. In some embodiments, theheterologous polypeptide is recovered from the host cell culture medium.In some embodiments, the method further comprises combining therecovered heterologous polypeptide (e.g., an antibody) with apharmaceutically acceptable carrier, excipient, or carrier to prepare apharmaceutical formulation comprising the heterologous polypeptide(e.g., antibody).

In one aspect, the invention provides methods of secreting aheterologous polypeptide of interest from a cell, said method comprisingculturing a host cell comprising a polynucleotide of the invention sothat the polynucleotide is expressed and the heterologous polypeptide issecreted.

In one aspect, the invention provides methods of translocating aheterologous polypeptide of interest from a cell, said method comprisingculturing a host cell comprising a polynucleotide of the invention sothat the polynucleotide is expressed and the heterologous polypeptide istranslocated.

In another aspect, the invention provides method of optimizing secretionof a heterologous polypeptide of interest in a cell comprising comparingthe levels of expression of the polypeptide under control of a set ofpolynucleotide variants of a translation initiation region, wherein theset of variants represents a range of translational strengths, anddetermining the optimal translational strength for production of maturepolypeptide. In some embodiments, the optimal translational strength isless than the translational strength of the wild-type translationinitiation region. In some embodiments, the optimal translationalstrength is more than the translational strength of the wild-typetranslation initiation region. In some embodiments, the variantscomprise polynucleotide variants of a secretion signal sequence. In someembodiments, the variant secretion signal sequences are sec pathwaysignal sequences and/or SRP pathway signal sequences. In someembodiments, the variant secretion signal sequences are PhoA, MalE,DsbA, or STII variant signal sequences. In some embodiments, the variantis one or more variant shown in Table 3. In some embodiments, thevariant comprises sequence of one of SEQ ID Nos 1-14, 36-39, 41-42.

In one aspect, the invention provides a heterologous polypeptideobtained by a method of the invention as described herein. In someembodiments, the heterologous polypeptide is an antibody.

In one aspect, the invention provides uses of a heterologous polypeptidegenerated using the methods of the invention, in the preparation of amedicament for the therapeutic and/or prophylactic treatment of adisease, such as a cancer, a tumor, a cell proliferative disorder,and/or an immune (such as autoimmune) disorder. The heterologouspolypeptide can be of any form described herein, including antibody,antibody fragment, polypeptide (e.g., an oligopeptide), or combinationthereof.

In one aspect, the invention provides use of a polynucleotide of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder and/or an immune (such as autoimmune) disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder and/or an immune (such as autoimmune) disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder and/or an immune (such as autoimmune) disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumor, acell proliferative disorder, an immune (such as autoimmune) disorderand/or an angiogenesis-related disorder (wound healing).

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor, a cell proliferativedisorder and/or an immune (such as autoimmune) disorder).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Translocation of indicated signal peptides across the innermembrane of bacteria. The maltose-binding periplasmic protein (MalE) andalkaline phosphatase (PhoA) signal peptides direct translocation fromthe cytoplasm to the periplasm in a post-translational manner with theaid of the molecular motor SecA. The heat-stable enterotoxin II (StII)and thiol:disulfide interchange protein (DsbA) signal peptides directtranslocation in a co-translational manner with aid from the signalrecognition particle (SRP).

FIG. 2: Relative translocation initiation region strength of signalpeptide variants. Normalized basal alkaline phosphatase activity of 27C7cells carrying a vector with a fusion between either an STII, MalE,PhoA, or DsbA signal peptide and the mature domain of the E. colialkaline phosphatase (BAP) gene. Each bar represents an individualculture incubated with the chromogenic substrate PNPP and enzymaticactivity was determined as the absorbance of that culture at 410 nm lessthe absorbance of a culture carrying an empty vector (pBR322).Activities were normalized to the basal activity of 27C7 cells carryingthe plasmid pPho41. White bars represent signal peptide variants with aBssHII restriction site at the −9 position relative to the first basepair of the initiation codon. Grey or striped bars represent an MluI orXbaI site at the −9 position, respectively. All activities are the meanof between seven and ten replicate experiments. Error bars are reportedas the uncertainty in the mean with a 95% confidence limit. Thedifferences in relative TIR strength between adjacent bars are allstatistically significant (P<<0.001). Bars represent clones SH1.2,SH2.41, SH3.38, SH4.60, SH5.34, SH6.52, SH8.36, SL1.2, SL2.74, SL3.72,MH1.92, MH2.100, ML1.97, ML2.123, MX1.wt, MX2.15, MX3.12, MX5.37, MX6.4,MX7.25, MX8.13, MX11.34, PH1.70, PH2.64, PH3.wt, PH4.67, PH5.71, PH6.77,PL1.104, PX2.41, PX3.wt, PX5.53, PX6.15, PX8.24, PX10.23, DH1.48,DH2.wt, DH3.79, DH7.72, DLl.wt, DL2.3, DL3.37 (in order, from left toright).

FIG. 3: Monitoring assembly of antibody species with heavy chain signalpeptide manipulation. 64B4 cells were grown in 25 mL of C.R.A.P.phosphate-limiting media for 24 hours and soluble fractions as well attotal protein pellets normalized by optical density (OD) were preparedfor SDS-PAGE analysis. (A) Samples from cells carrying the plasmidpBR-SS-5D5-1.1 (SS1.1), pBR-MS-5D5-1.1 (MS1.1), pBR-DS-5D5-1.1 (DS1.1)or pBR-PS-5D5-1.1 (PS1.1) were separated by SDS-PAGE gel electrophoresis(mass in kDa indicated at the left side), transferred to nitrocellulose,and probed for the presence of heavy chain-containing species with anα-Fc specific antibody. Soluble samples (top blot) consisted of theputatively identified bands corresponding to (from top to bottom):full-length antibody, heavy-heavy-light (HHL), heavy-light (HL) or freeheavy chain (heavy chain monomer). Normalized, total protein samples(bottom blot) were reduced with 0.2 M DTT to disrupt disulfide bondstructure and each individual lane migrated to a single band with anapparent mass of ˜49 kDa. (B) The samples from (A) were run on aseparate SDS-PAGE gel (mass in kDa indicated at the right side),transferred to nitrocellulose and probed for complexes containing alight chain with an α-κLc specific antibody. Soluble samples (top blot)consisted of the putatively identified bands corresponding to (from topto bottom): full-length antibody, HL, light-light (LL) dimer or freelight chain (light chain monomer). Normalized, total protein samples(bottom blot) were reduced with 0.2 M DTT and each individual lanemigrated to a single band with an apparent mass of ˜25 kDa.Abbreviations: S=signal sequence STII M=signal sequence MalE D=signalsequence DsbA P=signal sequence PhoA. XX#.# (e.g. DS1.1) refers to heavychain signal sequence, light chain signal sequence, heavy chain TIR,light chain TIR used in the experiment.

FIG. 4: Monitoring assembly of antibody species with light chain signalpeptide manipulation. 64B4 cells were grown in 25 mL of C.R.A.P.phosphate-limiting media for 24 hours and soluble fractions as well attotal protein pellets normalized by optical density (OD) were preparedfor SDS-PAGE analysis. Samples from cells carrying the plasmidpBR-DS-5D5-1.1 (DS1.1), pBR-DS-5D5-1.2 (DS1.2), pBR-DM-5D5-1.1 (DM1.1),pBR-DM-5D5-1.2 (DM1.2), pBR-DD-5D5-1.1 (DD1.1), pBR-DD-5D5-1.2 (DD1.2),pBR-DP-5D5-1.1 (DP1.1), or pBR-DP-5D5-1.2 were separated by SDS-PAGE gelelectrophoresis (mass in kDa indicated at the left side), transferred tonitrocellulose, and probed for the presence of heavy or lightchain-containing species with an α-Fc or α-κLc specific antibody,respectively, as indicated along the right side of the images. Solublesamples (top blot) consisted of the putatively identified bandscorresponding to (from top to bottom): full-length antibody,heavy-heavy-light (HHL), heavy-light (HL) dimer or free heavy chain.Normalized, total protein samples (middle blot, bottom) were reducedwith 0.2 M DTT to disrupt disulfide bond structure and each individuallane migrated to a single band with an apparent mass of ˜49 kDa whenprobed with an α-Fc antibody. When probed with an α-κLc specificantibody, all lanes migrated to a single or double band with an apparentmass of either ˜25 kDa or ˜27 kDa and ˜25 kDa. Abbreviations: S=signalsequence STII M=signal sequence MalE D=signal sequence DsbA P=signalsequence PhoA. XX#.# (e.g. DS1.1) refers to heavy chain signal sequence,light chain signal sequence, heavy chain TIR, light chain TIR used inthe experiment.

FIG. 5: Monitoring assembly of antibody species over time from 10-Lfermentations. 64B4 cells were grown to a high cell density in a 10-Lfermentation for three days with samples taken at regular time intervals(time sample taken above each lane in hours past inoculation) from whichsoluble fractions as well at total protein pellets normalized by opticaldensity (OD) were prepared for SDS-PAGE analysis. Samples from cellscarrying the plasmid pBR-SS-5D5-1.1 co-expressing the chaperone-bearingplasmid pJJ247 (SS1.1+Chaperones), pBR-DD-5D5-1.1 with pJJ247(DD1.1+Chaperones), pBR-DS-5D5-1.1 with pJJ247 (DM1.1+Chaperones), orpBR-DP-5D5-1.1 with pJJ247 (DP1.1+Chaperones) were separated by SDS-PAGEgel electrophoresis (mass in kDa indicated at the left side),transferred to nitrocellulose, and probed for the presence of heavy orlight chain-containing species with an α-Fc or α-κLc specific antibody,respectively, as indicated along the right side of the images. Solublesamples (top blot) consisted of the putatively identified bandscorresponding to (from top to bottom): full-length antibody,heavy-heavy-light (HHL), heavy-light (HL) dimer, light-light (LL) dimer,or free light chain. Normalized, total protein samples (middle blot,bottom) were reduced with 0.2 mM DTT to disrupt disulfide bond structureand each individual lane migrated to a single band with an apparent massof ˜49 kDa when probed with an α-Fc. When probed with an α-κLc specificantibody, all lanes migrated to a single band with an apparent mass ofeither ˜25 kDa. Abbreviations: S=signal sequence STII M=signal sequenceMalE D=signal sequence DsbA P=signal sequence PhoA. XX#.# (e.g. DS1.1)refers to heavy chain signal sequence, light chain signal sequence,heavy chain TIR, light chain TIR used in the experiment.

FIG. 6: Accumulation of mature PhoA under inducing conditions. 27C7cells were grown in 25-mL of C.R.A.P. phosphate-limiting media for 24hours and soluble fractions were normalized by optical density (OD) andprepared for SDS-PAGE analysis. The mature domain of the E. coli phoAgene was fused to the indicated DsbA or STII (bottom) TIR variants(top). Gel was visualized for the presence of protein by Commassie bluestaining A putatively identified band corresponding to the mature domainof PhoA (right) appeared at a mass of ˜47 kDa (mass indicated at leftside).

FIG. 7: depicts amino acid sequences of the framework (FR), CDR, firstconstant domain (CL or CH1) and Fc region (Fc) of MetMAb (OA5D5v2).Figure discloses Light Chain sequences as SEQ ID NOS 57-60, 49-51 & 61,respectively, in order of appearance and Heavy Chain sequences as SEQ IDNOS 62-65, 52-53 & 66-68, respectively, in order of appearance. The Fcsequence depicted comprises “hole” (cavity) mutations T366S, L368A andY407V, as described in WO 2005/063816.

FIG. 8: depicts sequence of an Fc polypeptide (SEQ ID NO: 47) comprising“knob” (protuberance) mutation T366W, as described in WO 2005/063816. Inone embodiment, an Fc polypeptide comprising this sequence forms acomplex with an Fc polypeptide comprising the Fc sequence of FIG. 7 togenerate an Fc region.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS 1NMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

The term “cistron,” as used herein, is intended to refer to a geneticelement broadly equivalent to a translational unit comprising thenucleotide sequence coding for a polypeptide chain and adjacent controlregions. “Adjacent control regions” include, for example, atranslational initiation region (TIR; as defined herein below) and atermination region.

A “polycistronic” expression vector refers to a single vector thatcontains and expresses multiple cistrons under the regulatory control ofone single promoter. A common example of polycistronic vector is a“dicistronic” vector that contains and expresses two differentpolypeptides under the control of one promoter. Upon expression of adicistronic or polycistronic vector, multiple genes are firsttranscribed as a single transcriptional unit, and then translatedseparately.

A “separate cistron” expression vector according to the presentinvention refers to a single vector comprising at least two separatepromoter-cistron pairs, wherein each cistron is under the control of itsown promoter. Upon expression of a separate cistron expression vector,both transcription and translation processes of different genes areseparate and independent.

The “translation initiation region” or TIR or translational initiationregion or translational initiation sequence, as used herein refers to anucleic acid region providing the efficiency of translational initiationof a gene of interest. In general, a TIR within a particular cistronencompasses the ribosome binding site (RBS) and sequences 5′ and 3′ toRBS. The RBS is defined to contain, minimally, the Shine-Dalgarno regionand the start codon (AUG). Accordingly, a TIR also includes at least aportion of the nucleic acid sequence to be translated. Preferably, a TIRof the invention includes a secretion signal sequence encoding a signalpeptide that precedes the sequence encoding for the light or heavy chainwithin a cistron. A TIR variant contains sequence variants (particularlysubstitutions) within the TIR region that alter the property of the TIR,such as its translational strength as defined herein below. Preferably,a TIR variant of the invention contains sequence substitutions withinthe first 2 to about 14, preferably about 4 to 12, more preferably about6 codons of the secretion signal sequence that precedes the sequenceencoding for the light or heavy chain within a cistron.

The term “translational strength” as used herein refers to a measurementof a secreted polypeptide in a control system wherein one or morevariants of a TIR is used to direct secretion of a polypeptide and theresults compared to the wild-type TIR or some other control under thesame culture and assay conditions. Without being limited to any onetheory, “translational strength” as used herein can include, forexample, a measure of mRNA stability, efficiency of ribosome binding tothe ribosome binding site, and mode of translocation across a membrane.

“Secretion signal sequence” or “signal sequence” refers to a nucleicacid sequence encoding for a short signal peptide that can be used todirect a newly synthesized protein of interest through a cellularmembrane, usually the inner membrane or both inner and outer membranesof prokaryotes. As such, the protein of interest such as theimmunoglobulin light or heavy chain polypeptide is secreted into theperiplasm of the prokaryotic host cells or into the culture medium. Thesignal peptide encoded by the secretion signal sequence may beendogenous to the host cells, or they may be exogenous, including signalpeptides native to the polypeptide to be expressed. Secretion signalsequences are typically present at the amino terminus of a polypeptideto be expressed, and are typically removed enzymatically betweenbiosynthesis and secretion of the polypeptide from the cytoplasm. Thus,the signal peptide is usually not present in a mature protein product.

“Operably linked” refers to a juxtaposition of two or more components,wherein the components so described are in a relationship permittingthem to function in their intended manner. For example, a promoter isoperably linked to a coding sequence if it acts in cis to control ormodulate the transcription of the linked sequence. Generally, but notnecessarily, the DNA sequences that are “operably linked” are contiguousand, where necessary to join two protein coding regions or in the caseof a secretory leader, contiguous and in reading frame. However,although an operably linked promoter is generally located upstream ofthe coding sequence, it is not necessarily contiguous with it. Operablylinked enhancers can be located upstream, within or downstream of codingsequences and at considerable distances from the promoter. Linking isaccomplished by recombinant methods known in the art, e.g., using PCRmethodology, by annealing, or by ligation at convenient restrictionsites. If convenient restriction sites do not exist, then syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

“Regulatory elements” as used herein, refer to nucleotide sequencespresent in cis, necessary for transcription and translation of apolynucleotide encoding a heterologous polypeptide into polypeptides.The transcriptional regulatory elements normally comprise a promoter 5′of the gene sequence to be expressed, transcriptional initiation andtermination sites, and polyadenylation signal sequence. The term“transcriptional initiation site” refers to the nucleic acid in theconstruct corresponding to the first nucleic acid incorporated into theprimary transcript, i.e., the mRNA precursor; the transcriptionalinitiation site may overlap with the promoter sequences.

A “promoter” refers to a polynucleotide sequence that controlstranscription of a gene or sequence to which it is operably linked. Apromoter includes signals for RNA polymerase binding and transcriptioninitiation. The promoters used will be functional in the cell type ofthe host cell in which expression of the selected sequence iscontemplated. A large number of promoters including constitutive,inducible and repressible promoters from a variety of different sources,are well known in the art (and identified in databases such as GenBank)and are available as or within cloned polynucleotides (from, e.g.,depositories such as ATCC as well as other commercial or individualsources). With inducible promoters, the activity of the promoterincreases or decreases in response to a signal.

The term “host cell” (or “recombinant host cell”), as used herein, isintended to refer to a cell that has been genetically altered, or iscapable of being genetically altered by introduction of an exogenouspolynucleotide, such as a recombinant plasmid or vector. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

An “isolated” polypeptide (e.g., an antibody) is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, thepolypeptide will be purified (1) to greater than 95% by weight ofpolypeptide as determined by the Lowry method, and most preferably morethan 99% by weight, (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the nucleic acid (for example, an antibody encoding nucleicacid) where, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

As used herein, “polypeptide” refers generally to peptides and proteinsfrom any cell source having more than about ten amino acids.“Heterologous” polypeptides are those polypeptides foreign to the hostcell being utilized, such as a human protein produced by E. coli. Whilethe heterologous polypeptide may be prokaryotic or eukaryotic,preferably it is eukaryotic, more preferably mammalian, and mostpreferably human. Preferably, it is a recombinantly produced, orrecombinant polypeptide. “Heterologous” polypeptides are thosepolypeptides foreign to the host cell being utilized, such as a humanprotein produced by E. coli. While the heterologous polypeptide may beprokaryotic or eukaryotic, preferably it is eukaryotic, more preferablymammalian, and most preferably human. Preferably, it is a recombinantlyproduced, or recombinant polypeptide.

Examples of mammalian polypeptides include molecules such as, e.g.,renin, a growth hormone, including human growth hormone; bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; 1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; thrombopoietin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial naturieticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA) and variantsthereof such as RETEVASE™ and TNKASE™; bombesin; thrombin; hemopoieticgrowth factor; tumor necrosis factor-alpha and -beta; antibodies toErbB2 domain(s) such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697),which binds to a region in the extracellular domain of ErbB2 (e.g., anyone or more residues in the region from about residue 22 to aboutresidue 584 of ErbB2, inclusive), enkephalinase; a serum albumin such ashuman serum albumin; Muellerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; DNase; inhibin; activin;vascular endothelial growth factor (VEGF); receptors for hormones orgrowth factors; integrin; protein A or D; rheumatoid factors; aneurotrophic factor such as brain-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF; cardiotrophins (cardiac hypertrophy factor)such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(I-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD-3,CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; serum albumin, such as human serumalbumin (HSA) or bovine serum albumin (BSA); colony stimulating factors(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1to IL-10; anti-HER-2 antibody; Apo2 ligand; superoxide dismutase; T-cellreceptors; surface membrane proteins; decay accelerating factor; viralantigen such as, for example, a portion of the AIDS envelope; transportproteins; homing receptors; addressins; regulatory proteins; antibodies;and fragments of any of the above-listed polypeptides.

Preferred polypeptides herein include human serum albumin (HSA), 2C4,tissue factor, anti-tissue factor, anti-CD20, anti-HER-2, heregulin,anti-IgE, anti-CD11a, anti-CD18, VEGF and receptors and antibodiesthereto such as rhuFab V2 and AVASTINT™, growth hormone and itsvariants, such as hGH, growth hormone receptors, growth hormonereleasing protein (GHRP), LIV-1 (EP 1,263,780), TRAIL, tumor necrosisfactor (TNF) and antibodies thereto, TNF receptor and relatedantibodies, TNF-receptor-IgG, TNF receptor associated factors (TRAFs)and inhibitors thereof, Factor VIII, Factor VIII B domain, interferonssuch as interferon-gamma, transforming growth factors (TGFs) such asTGF-beta, anti-TGF such as anti-TGF-beta, activin, inhibin,anti-activin, anti-inhibin, tissue-plasminogen activators and theirvariants such as t-PA, RETEPLASE™, and TNKase, anti-Fas antibodies,Apo-2 ligand; Apo-2 ligand inhibitor; Apo-2 receptor, Apo-3, apoptoticfactors, Ced-4, DcR3, death receptor and agonist antibodies (DR4, DR5),lymphotoxin (LT), prolactin, prolactin receptor, SOB proteins, WISP(wnt-induced secreted proteins), neurotoxin-3 (NT-3), nerve growthfactor (NGF) and anti-NGF, DNase, hepatitis antigen, herpes simplexantigen, leptin, insulin-like growth factors (IGFs) such as IGF-1 andIGF-2 and their binding proteins and receptors such as IGFBP-1-IGFBP-6,insulin, fibroblast growth factors (FGFs) such as FGF-17, Toll protein,TIE ligands, CD40 and anti-CD40, immunoadhesins, subtilisin, hepatocytegrowth factor (HGF), thrombopoietin (TPO), interleukins such as IL-2,IL-12, IL-17, IL-22, IL-8, IL-9, and antibodies thereto, andprostrate-specific cancer antigen (PSCA).

Particularly preferred polypeptides are recombinant polypeptides, morepreferably antibodies, which include monoclonal antibodies and humanizedantibodies. Such antibodies may be full-length antibodies or antibodyfragments. More preferably, these antibodies are human or humanizedantibodies. Still more preferably, the antibody is an anti-c-met,anti-IgE, anti-CD18, anti-VEGF, anti-tissue factor, 2C4, anti-Her-2,anti-CD20, anti-CD40, or anti-CD11 a antibody. Antibody fragmentsencompassed within the definition of polypeptide preferably comprise alight chain, more preferably a kappa light chain. Such preferredfragments include, for example, a Fab, Fab′, F(ab′)₂, or F(ab′)₂-leucinezipper (LZ) fusion, and a one-armed antibody.

Protein “expression” refers to conversion of the information encoded ina gene into messenger RNA (mRNA) and then to the protein.

An “immunoconjugate” (interchangeably referred to as “antibody-drugconjugate,” or “ADC”) means an antibody conjugated to one or morecytotoxic agents, such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., a protein toxin, an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

A “blocking” antibody or an antibody “antagonist” is one which inhibitsor reduces biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest(e.g., HGF).

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Desirably the Kd is 1×10⁻⁷, 1×10⁻⁸, 5×10⁻⁸,1×10⁻⁹, 3×10⁻⁹, 5×10⁻⁹, or even 1×10⁻¹⁰ or stronger. Affinity can bemeasured by common methods known in the art, including those describedherein. Low-affinity antibodies generally bind antigen slowly and tendto dissociate readily, whereas high-affinity antibodies generally bindantigen faster and tend to remain bound longer. A variety of methods ofmeasuring binding affinity are known in the art, any of which can beused for purposes of the present invention. Specific illustrativeembodiments are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate ofSW/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Insome embodiments, the following modifications are used for the surfacePlasmon resonance assay method: antibody is immobilized to CMS biosensorchips to achieve approximately 400 RU, and for kinetic measurements,two-fold serial dilutions of target protein are injected in PBST bufferat 25° C. with a flow rate of about 30 ul/minute. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) is calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol. 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CMS chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 uM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Insome embodiments, the following modifications are used for the surfacePlasmon resonance assay method: antibody is immobilized to CM5 biosensorchips to achieve approximately 400 RU, and for kinetic measurements,two-fold serial dilutions of target protein are injected in PBST bufferat 25° C. with a flow rate of about 30 ul/minute. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol. 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

A “naked antibody” is an antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety or radiolabel.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences of this invention, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequenceof this invention so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence of this invention. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie et al., Ann. Rev. Immunol. 18:739-766 (2000), Table 1).Antibodies with substitutions in an Fc region thereof and increasedserum half-lives are also described in WO00/42072, WO 02/060919; Shieldset al., J. Biol. Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem.279:6213-6216 (2004)). In another embodiment, the serum half-life canalso be increased, for example, by attaching other polypeptidesequences. For example, antibodies or other polypeptides useful in themethods of the invention can be attached to serum albumin or a portionof serum albumin that binds to the FcRn receptor or a serum albuminbinding peptide so that serum albumin binds to the antibody orpolypeptide, e.g., such polypeptide sequences are disclosed inWO01/45746. In one preferred embodiment, the serum albumin peptide to beattached comprises an amino acid sequence of DICLPRWGCLW (SEQ ID NO:48). In another embodiment, the half-life of a Fab is increased by thesemethods. See also, Dennis et al. J. Biol. Chem. 277:35035-35043 (2002)for serum albumin binding peptide sequences.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule that contains, preferably, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or morenucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,180, 190, 200 amino acids or more.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity) and may alsoinclude certain antibody fragments (as described in greater detailherein). An antibody can be human, humanized, and/or affinity matured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. “Antibody fragments” comprise only aportion of an intact antibody, wherein the portion preferably retains atleast one, preferably most or all, of the functions normally associatedwith that portion when present in an intact antibody. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. In oneembodiment, an antibody fragment comprises an antigen binding site ofthe intact antibody and thus retains the ability to bind antigen. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function andcomplement binding. In one embodiment, an antibody fragment is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For e.g., such an antibody fragment may compriseon antigen binding arm linked to an Fc sequence capable of conferring invivo stability to the fragment.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al., Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins. WO 00/42072 (Presta) describes antibody variants withimproved or diminished binding to FcRs. The content of that patentpublication is specifically incorporated herein by reference. See, also,Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO 99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The term “Fc region”, as used herein, generally refers to a dimercomplex comprising the C-terminal polypeptide sequences of animmunoglobulin heavy chain, wherein a C-terminal polypeptide sequence isthat which is obtainable by papain digestion of an intact antibody. TheFc region may comprise native or variant Fc sequences. Although theboundaries of the Fc sequence of an immunoglobulin heavy chain mightvary, the human IgG heavy chain Fc sequence is usually defined tostretch from an amino acid residue at about position Cys226, or fromabout position Pro230, to the carboxyl terminus of the Fc sequence. TheFc sequence of an immunoglobulin generally comprises two constantdomains, a CH2 domain and a CH3 domain, and optionally comprises a CH4domain. By “Fc polypeptide” herein is meant one of the polypeptides thatmake up an Fc region. An Fc polypeptide may be obtained from anysuitable immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes,IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprisespart or all of a wild type hinge sequence (generally at its N terminus).In some embodiments, an Fc polypeptide does not comprise a functional orwild type hinge sequence.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues of the species-dependent antibody have beenmodified. Such mutants necessarily have less than 100% sequence identityor similarity with the species-dependent antibody. In one embodiment,the antibody mutant will have an amino acid sequence having at least 75%amino acid sequence identity or similarity with the amino acid sequenceof either the heavy or light chain variable domain of thespecies-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e. same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadyhaving a benign, pre-cancerous, or non-metastatic tumor as well as thosein which the occurrence or recurrence of cancer is to be prevented.

The term “therapeutically effective amount” refers to an amount of atherapeutic agent to treat or prevent a disease or disorder in a mammal.In the case of cancers, the therapeutically effective amount of thetherapeutic agent may reduce the number of cancer cells; reduce theprimary tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, time to diseaseprogression (TTP), the response rates (RR), duration of response, and/orquality of life.

An “autoimmune disease” herein is a non-malignant disease or disorderarising from and directed against an individual's own tissues. Theautoimmune diseases herein specifically exclude malignant or cancerousdiseases or conditions, especially excluding B cell lymphoma, acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia and chronic myeloblastic leukemia. Examples of autoimmunediseases or disorders include, but are not limited to, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); respiratory distress syndrome(including adult respiratory distress syndrome; ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g. Type Idiabetes mellitus or insulin dependent diabetes mellitis); multiplesclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder; multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers. By “early stage cancer” or “early stage tumor” ismeant a cancer that is not invasive or metastatic or is classified as aStage 0, I, or II cancer. Examples of cancer include, but are notlimited to, carcinoma, lymphoma, blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer (including metastatic breast cancer),colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer,tumors of the biliary tract, as well as head and neck cancer andmultiple myeloma.

The term “pre-cancerous” refers to a condition or a growth thattypically precedes or develops into a cancer. A “pre-cancerous” growthwill have cells that are characterized by abnormal cell cycleregulation, proliferation, or differentiation, which can be determinedby markers of cell cycle regulation, cellular proliferation, ordifferentiation.

By “dysplasia” is meant any abnormal growth or development of tissue,organ, or cells. Preferably, the dysplasia is high grade orprecancerous.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass.

Both stimulatory and inhibitory molecular pathways within the tumor cellregulate this behavior, and interactions between the tumor cell and hostcells in the distant site are also significant.

By “non-metastatic” is meant a cancer that is benign or that remains atthe primary site and has not penetrated into the lymphatic or bloodvessel system or to tissues other than the primary site. Generally, anon-metastatic cancer is any cancer that is a Stage 0, I, or II cancer,and occasionally a Stage III cancer.

By “primary tumor” or “primary cancer” is meant the original cancer andnot a metastatic lesion located in another tissue, organ, or location inthe subject's body.

By “benign tumor” or “benign cancer” is meant a tumor that remainslocalized at the site of origin and does not have the capacity toinfiltrate, invade, or metastasize to a distant site.

By “tumor burden” is meant the number of cancer cells, the size of atumor, or the amount of cancer in the body. Tumor burden is alsoreferred to as tumor load.

By “tumor number” is meant the number of tumors.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Preferably, the subject is a human.

The term “anti-cancer therapy” refers to a therapy useful in treatingcancer. Examples of anti-cancer therapeutic agents include, but arelimited to, e.g., chemotherapeutic agents, growth inhibitory agents,cytotoxic agents, agents used in radiation therapy, anti-angiogenesisagents, apoptotic agents, anti-tubulin agents, and other agents to treatcancer, anti-CD20 antibodies, platelet derived growth factor inhibitors(e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,celecoxib), interferons, cytokines, antagonists (e.g., neutralizingantibodies) that bind to one or more of the following targets ErbB2,ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s),TRAIL/Apo2, and other bioactive and organic chemical agents, etc.Combinations thereof are also included in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimeterxate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)(including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;VELCADE bortezomib; REVLIMID lenalidomide; leucovorin (LV); oxaliplatin,including the oxaliplatin treatment regimen (FOLFOX); inhibitors ofPKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva™)) and VEGF-A thatreduce cell proliferation and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON•toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids or derivativesof any of the above.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

A “biologically active” or “functional” polypeptide (such as aheterologous polypeptide) is one capable of exerting one or more of itsnatural activities in structural, regulatory, biochemical or biophysicalevents.

A “biologically active” or “functional” antibody is one capable ofexerting one or more of its natural activities in structural,regulatory, biochemical or biophysical events. For example, abiologically active antibody may have the ability to specifically bindan antigen and the binding may in turn elicit or alter a cellular ormolecular event such as signaling transduction or enzymatic activity. Abiologically active antibody may also block ligand activation of areceptor or act as an agonist antibody. The capability of a antibody toexert one or more of its natural activities depends on several factors,including proper folding and assembly of the polypeptide chains. As usedherein, the biologically active antibody generated by the disclosedmethods typically comprise heterotetramers having two identical L chainsand two identical H chains that are linked by multiple disulfide bondsand properly folded.

Compositions of the Invention and Methods Using Same

In one aspect, the present invention provides TIR variants. Thus, for agiven TIR, a series of amino acid or nucleic acid sequence variants canbe created with a range of translational strengths, thereby providing aconvenient means by which to adjust this factor for the optimalsecretion of many different polypeptides. The use of a reporter geneexpressed under the control of these variants, such as PhoA, provides amethod to quantitate the relative translational strengths of differenttranslation initiation regions. The variant or mutant TIRs can beprovided in the background of a plasmid vector thereby providing a setof plasmids into which a gene of interest may be inserted and itsexpression measured, so as to establish an optimum range oftranslational strengths for maximal expression of mature polypeptide.

Mutagenesis of the TIR is done by conventional techniques that result incodon changes which can alter the amino acid sequence, although silentchanges in the nucleotide sequence are preferred. Alterations in the TIRcan include, for example, alterations in the number or spacing ofShine-Dalgarno sequences, along with alterations in the signal sequence.One method for generating mutant signal sequences is the generation of a“codon bank” at the beginning of a coding sequence that does not changethe amino acid sequence of the signal sequence (i.e., the changes aresilent). This can be accomplished by changing the third nucleotideposition of each codon; additionally, some amino acids, such as leucine,serine, and arginine, have multiple first and second positions that canadd complexity in making the bank. This method of mutagenesis isdescribed in detail in Yansura et al. (METHODS: A Companion to Methodsin Enzymol. 4:151-158 (1992)). Basically, a DNA fragment encoding thesignal sequence and the beginning of the mature polypeptide issynthesized such that the third (and, possibly, the first and second, asdescribed above) position of each of the first 6 to 12 codons isaltered. The additional nucleotides downstream of these codons provide asite for the binding of a complementary primer used in making the bottomstrand. Treatment of the top coding strand and the bottom strand primerwith DNA polymerase I (Klenow) will result in a set of duplex DNAfragments containing randomized codons. The primers are designed tocontain useful cloning sites that can then be used to insert the DNAfragments in an appropriate vector, thereby allowing amplification ofthe codon bank. Alternative methods include, for example, replacement ofthe entire rbs with random nucleotides (Wilson et al., BioTechniques17:944-952 (1994)), and the use of phage display libraries (see, forexample, Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 89:4457-4461(1992); Garrard et al., Gene 128:103-109 (1993)).

The bacterial Sec translocase facilitates protein export in prokaryotes.Secretory proteins can be targeted to the Sec translocase by twodifferent mechanisms, ie, the co-translational and thepost-translational targeting. In the latter, the signal sequencecontaining secretory protein is released from the ribosome in itssynthesis completed state and directed to the Sec-translocase. Invarious Gram-negative bacteria, secretory proteins are guided to theSec-translocase by the secretion specific chaperone SecB that maintainsthese proteins in a translocation-competent, unfolded state. Duringco-translational targeting, the signal recognition particle (SRP) bindsto the signal sequence of the secretory protein while it emerges fromthe ribosome and the entire ternary complex of SRP/ribosome/nascentsecretory protein chain is targeted to the Sec-translocase.

For example, the maltose-binding periplasmic protein (MalE) and alkalinephosphatase (PhoA) signal peptides direct translocation from thecytoplasm to the periplasm in a post-translational manner with the aidof the molecular motor SecA. Other exemplary signal peptides that directtranslocation in a post-translational manner are dsbC, lolA, ompA, lamb,and lpp. The heat-stable enterotoxin II (stII) and thiol:disulfideinterchange protein (dsbA) signal peptides direct translocation in aco-translational manner with aid from the signal recognition particle(SRP). Other exemplary signal peptides that direct translocation in aco-translational manner are yraI, tort, tolB, sfmC, nikA, and sfmC. Seealso Natale et al. for a review of Sec- and Tat-mediated proteinsecretion across the bacterial cytoplasmic membrane. (Natale et al.(2008) Biochemica et Biophysica Acta 1778:1735-56.)

We developed novel variant translational initiation region (TIR) signalpeptide libraries (FIG. 2, Table 2) for signal peptides representing twoof the major secretion pathways for transport across the inner-membranein E. coli: sec (PhoA, MalE) and SRP (DsbA, STII). Each librarycomprises a panel of vectors with comprising variant TIRs of differingtranslational strengths, providing a means by which to readily adjustlevel of translation for a given protein of interest.

Typically, the TIR variants will be provided in a plasmid vector withappropriate elements for expression of a gene of interest. For example,a typical construct will contain a promoter 5′ to the signal sequence, arestriction enzyme recognition site 3′ to the signal sequence forinsertion of a gene of interest or a reporter gene, and a selectablemarker, such as a drug resistance marker, for selection and/ormaintenance of bacteria transformed with the resulting plasmids. Plasmidvectors are further discussed and exemplified herein. Promoters suitablefor use with prokaryotic hosts are known in the art and some areexemplified and described herein.

Any reporter gene may be used which can be quantified in some manner.Thus, for example, alkaline phosphatase production can be quantitated asa measure of the secreted level of the phoA gene product. Other examplesinclude, for example, the β-lactamase genes.

Generally, a set of vectors may be generated with a range of TIRstrengths for each cistron of the vector therein. This limited setprovides a comparison of expression levels of each chain as well as theyield of full length products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. For the purpose of this invention, the translational strengthcombination for a particular pair of TIRs within a vector is representedby (N-light, M-heavy), wherein N is the relative TIR strength of lightchain and M is the relative TIR strength of heavy chain. For example,(3-light, 7-heavy) means the vector provides a relative TIR strength ofabout 3 for light chain expression and a relative TIR strength of about7 for heavy chain expression. Based on the translational strengthcomparison, the desired individual TIRs are selected to be combined inthe expression vector constructs of the invention. Vectors soconstructed can be used to transform an appropriate host. Preferably,the host is a prokaryotic host. More preferably, the host is E. coli.

The secreted level of polypeptides can be determined, for example, by afunctional assays for the polypeptide of interest, if available,radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), or by PAGEand visualization of the correct molecular weight of the polypeptide ofinterest. Methods for determining level of secreted polypeptide are wellknown in the art and some are exemplified herein.

Antibodies

The antibodies of the invention are preferably monoclonal. Alsoencompassed within the scope of the invention are Fab, Fab′, Fab′-SH andF(ab′)₂ fragments of the antibodies provided herein. These antibodyfragments can be created by traditional means, such as enzymaticdigestion, or may be generated by recombinant techniques. Such antibodyfragments may be chimeric or humanized. These fragments are useful forthe diagnostic and therapeutic purposes set forth below.

Accordingly, in some embodiment, the anti-c-met antibody is a one-armedantibody (i.e., the heavy chain variable domain and the light chainvariable domain form a single antigen binding arm) comprising an Fcregion, wherein the Fc region comprises a first and a second Fcpolypeptide, wherein the first and second Fc polypeptides are present ina complex and form a Fc region that increases stability of said antibodyfragment compared to a Fab molecule comprising said antigen binding arm.For treatment of pathological conditions requiring an antagonisticfunction, and where bivalency of an antibody results in an undesirableagonistic effect, the monovalent trait of a one-armed antibody (i.e., anantibody comprising a single antigen binding arm) results in and/orensures an antagonistic function upon binding of the antibody to atarget molecule. Furthermore, the one-armed antibody comprising a Fcregion is characterized by superior pharmacokinetic attributes (such asan enhanced half life and/or reduced clearance rate in vivo) compared toFab forms having similar/substantially identical antigen bindingcharacteristics, thus overcoming a major drawback in the use ofconventional monovalent Fab antibodies. One-armed antibodies aredisclosed in, for example, WO2005/063816; Martens et al, Clin Cancer Res(2006), 12: 6144. In some embodiments, the one armed antibody is amonovalent antibody fragment, wherein the antibody fragment comprises afirst polypeptide comprising a light chain variable domain, a secondpolypeptide comprising a heavy chain variable domain and said first Fcpolypeptide, and a third polypeptide comprising said second Fcpolypeptide, whereby the heavy chain variable domain and the light chainvariable domain form a single antigen binding arm, and whereby the firstand second Fc polypeptides are present in a complex and form a Fc regionthat increases stability of said antibody fragment compared to a Fabmolecule comprising said antigen binding arm.

In some embodiments, the antibody binds (in some embodiments,specifically binds) c-met. In some embodiments, the anti-c-met antibodycomprises (a) a first polypeptide comprising a heavy chain variabledomain having the sequence:EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS(SEQ ID NO: 43), CH1 sequence, and a first Fc polypeptide; (b) a secondpolypeptide comprising a light chain variable domain having thesequence:DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR(SEQ ID NO: 44), and CL1 sequence; and (c) a third polypeptidecomprising a second Fc polypeptide, wherein the heavy chain variabledomain and the light chain variable domain are present as a complex andform a single antigen binding arm, wherein the first and second Fcpolypeptides are present in a complex and form a Fc region thatincreases stability of said antibody fragment compared to a Fab moleculecomprising said antigen binding arm. In some embodiments, the firstpolypeptide comprises the Fc sequence depicted in FIG. 7 (SEQ ID NO: 68)and the second polypeptide comprises the Fc sequence depicted in FIG. 8(SEQ ID NO: 47). In some embodiments, the first polypeptide comprisesthe Fc sequence depicted in FIG. 8 (SEQ ID NO: 47) and the secondpolypeptide comprises the Fc sequence depicted in FIG. 7 (SEQ ID NO:68).

In some embodiments, the anti-c-met antibody comprises (a) a firstpolypeptide comprising a heavy chain variable domain, said polypeptidecomprising the sequence:EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 45); (b) a second polypeptide comprising a light chain variabledomain, the polypeptide comprising the sequenceDIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 46); and a third polypeptide comprising a Fc sequence, thepolypeptide comprising the sequenceDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 47), wherein the heavy chain variable domain and the lightchain variable domain are present as a complex and form a single antigenbinding arm, wherein the first and second Fc polypeptides are present ina complex and form a Fc region that increases stability of said antibodyfragment compared to a Fab molecule comprising said antigen binding arm.

In one aspect, the anti-c-met antibody comprises:

(a) at least one, two, three, four or five hypervariable region (CDR)sequences selected from the group consisting of:

(i) CDR-L1 comprising sequence A1-A17, wherein A1-A17 isKSSQSLLYTSSQKNYLA (SEQ ID NO:49)

(ii) CDR-L2 comprising sequence B1-B7, wherein B1-B7 is WASTRES (SEQ IDNO:50)

(iii) CDR-L3 comprising sequence C1-C9, wherein C1-C9 is QQYYAYPWT (SEQID NO:51)

(iv) CDR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWLH(SEQ ID NO:52)

(v) CDR-H2 comprising sequence E1-E 18, wherein E1-E 18 isGMIDPSNSDTRFNPNFKD (SEQ ID NO:53) and

(vi) CDR-H3 comprising sequence F1-F11, wherein F1-F11 is T/SYGSYVSPLDY(SEQ ID NO:54);

and (b) at least one variant CDR, wherein the variant CDR sequencecomprises modification of at least one residue of the sequence depictedin (i)-(vi). In one embodiment, CDR-H3 comprises TYGSYVSPLDY (SEQ ID NO:55). In one embodiment, CDR-H3 comprises SYGSYVSPLDY (SEQ ID NO: 56). Inone embodiment, an antibody of the invention comprising these sequences(in combination as described herein) is humanized or human.

In one embodiment, the anti-c-met antibody comprises a heavy chainvariable domain comprising one or more of CDR1-HC, CDR2-HC and CDR3-HCsequence depicted in FIG. 7 (SEQ ID NO: 52-53 & 66). In someembodiments, the antibody comprises a light chain variable domaincomprising one or more of CDR1-LC, CDR2-LC and CDR3-LC sequence depictedin FIG. 7 (SEQ ID NOs: 49-51). In some embodiments, the heavy chainvariable domain comprises FR1-HC, FR2-HC, FR3-HC and FR4-HC sequencedepicted in FIG. 7 (SEQ ID NOs: 62-65). In some embodiments, the lightchain variable domain comprises FR1-LC, FR2-LC, FR3-LC and FR4-LCsequence depicted in FIG. 7 (SEQ ID NOs: 57-60).

Variant HVRs in an anti-c-met antibody of the invention can havemodifications of one or more residues within the HVR. In one embodiment,a HVR-L2 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions in anycombination of the following positions: B1 (M or L), B2 (P, T, G or S),B3 (N, G, R or T), B4 (I, N or F), B5 (P, I, L or G), B6 (A, D, T or V)and B7 (R, I, M or G). In one embodiment, a HVR-H1 variant comprises 1-5(1, 2, 3, 4 or 5) substitutions in any combination of the followingpositions: D3 (N, P, L, S, A, I), D5 (I, S or Y), D6 (G, D, T, K, R), D7(F, H, R, S, T or V) and D9 (M or V). In one embodiment, a HVR-H2variant comprises 1-4 (1, 2, 3 or 4) substitutions in any combination ofthe following positions: E7 (Y), E9 (I), E10 (I), E14 (T or Q), E15 (D,K, S, T or V), E16 (L), E17 (E, H, N or D) and E18 (Y, E or H). In oneembodiment, a HVR-H3 variant comprises 1-5 (1, 2, 3, 4 or 5)substitutions in any combination of the following positions: F1 (T, S),F3 (R, S, H, T, A, K), F4 (G), F6 (R, F, M, T, E, K, A, L, W), F7 (L, I,T, R, K, V), F8 (S, A), F10 (Y, N) and F11 (Q, S, H, F). Letter(s) inparenthesis following each position indicates an illustrativesubstitution (i.e., replacement) amino acid; as would be evident to oneskilled in the art, suitability of other amino acids as substitutionamino acids in the context described herein can be routinely assessedusing techniques known in the art and/or described herein. In oneembodiment, a HVR-L1 comprises the sequence of SEQ ID NO:49. In oneembodiment, F1 in a variant HVR-H3 is T. In one embodiment, F1 in avariant HVR-H3 is S. In one embodiment, F3 in a variant HVR-H3 is R. Inone embodiment, F3 in a variant HVR-H3 is S. In one embodiment, F7 in avariant HVR-H3 is T. In one embodiment, an antibody of the inventioncomprises a variant HVR-H3 wherein F1 is T or S, F3 is R or S, and F7 isT.

In one embodiment, an anti-c-met antibody of the invention comprises avariant HVR-H3 wherein F1 is T, F3 is R and F7 is T. In one embodiment,an antibody of the invention comprises a variant HVR-H3 wherein F1 is S.In one embodiment, an antibody of the invention comprises a variantHVR-H3 wherein F1 is T, and F3 is R. In one embodiment, an antibody ofthe invention comprises a variant HVR-H3 wherein F1 is S, F3 is R and F7is T. In one embodiment, an antibody of the invention comprises avariant HVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is S. In oneembodiment, an antibody of the invention comprises a variant HVR-H3wherein F1 is T, F3 is S, F7 is T, and F8 is A. In some embodiments,said variant HVR-H3 antibody further comprises HVR-L1, HVR-L2, HVR-L3,HVR-H1 and HVR-H2 wherein each comprises, in order, the sequencedepicted in SEQ ID NOs:49, 50, 51, 52, and 53. In some embodiments,these antibodies further comprise a human subgroup III heavy chainframework consensus sequence. In one embodiment of these antibodies, theframework consensus sequence comprises substitution at position 71, 73and/or 78. In some embodiments of these antibodies, position 71 is A, 73is T and/or 78 is A. In one embodiment of these antibodies, theseantibodies further comprise a human κI light chain framework consensussequence.

In one embodiment, an anti-c-met antibody of the invention comprises avariant HVR-L2 wherein B6 is V. In some embodiments, said variant HVR-L2antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,wherein each comprises, in order, the sequence depicted in SEQ ID NOs:49, 51, 52, 53, 54. In some embodiments, said variant HVR-L2 antibodyfurther comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, whereineach comprises, in order, the sequence depicted in SEQ ID NOs: 49, 51,52, 53, 55. In some embodiments, said variant HVR-L2 antibody furthercomprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs: 49, 51, 52,53, 56. In some embodiments, these antibodies further comprise a humansubgroup III heavy chain framework consensus sequence. In one embodimentof these antibodies, the framework consensus sequence comprisessubstitution at position 71, 73 and/or 78. In some embodiments of theseantibodies, position 71 is A, 73 is T and/or 78 is A. In one embodimentof these antibodies, these antibodies further comprise a human κI lightchain framework consensus sequence.

In one embodiment, an anti-cmet antibody of the invention comprises avariant HVR-H2 wherein E14 is T, E15 is K and E17 is E. In oneembodiment, an antibody of the invention comprises a variant HVR-H2wherein E17 is E. In some embodiments, said variant HVR-H3 antibodyfurther comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3 whereineach comprises, in order, the sequence depicted in SEQ ID NOs: 49, 50,51, 52, 54. In some embodiments, said variant HVR-H2 antibody furthercomprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs: 49, 50, 51,52, 55. In some embodiments, said variant HVR-H2 antibody furthercomprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs: 49, 50, 51,52, 55. In some embodiments, these antibodies further comprise a humansubgroup III heavy chain framework consensus sequence. In one embodimentof these antibodies, the framework consensus sequence comprisessubstitution at position 71, 73 and/or 78. In some embodiments of theseantibodies, position 71 is A, 73 is T and/or 78 is A. In one embodimentof these antibodies, these antibodies further comprise a human κI lightchain framework consensus sequence.

Other anti-c-met antibodies suitable for use in the methods of theinvention are known in the art.

In one aspect, the anti-c-met antibody comprises at least onecharacteristic that promotes heterodimerization, while minimizinghomodimerization, of the Fc sequences within the antibody fragment. Suchcharacteristic(s) improves yield and/or purity and/or homogeneity of theimmunoglobulin populations. In one embodiment, the antibody comprises Fcmutations constituting “knobs” and “holes” as described inWO2005/063816. For example, a hole mutation can be one or more of T366A,L368A and/or Y407V in an Fc polypeptide, and a knob mutation can beT366W. Knob and hole Fc mutations are further described herein.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The monoclonal antibodies of the invention can be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to antigen may be raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof antigen and an adjuvant. Antigen may be prepared using methodswell-known in the art, some of which are further described herein. Forexample, recombinant production of human and mouse antigen is describedbelow. In one embodiment, animals are immunized with a antigen fused tothe Fc portion of an immunoglobulin heavy chain. In a preferredembodiment, animals are immunized with a antigen-IgG1 fusion protein.Animals ordinarily are immunized against immunogenic conjugates orderivatives of antigen with monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.)and the solution is injected intradermally at multiple sites. Two weekslater the animals are boosted. 7 to 14 days later animals are bled andthe serum is assayed for antibody titer. Animals are boosted until titerplateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The antibodies of the invention can be made by using combinatoriallibraries to screen for synthetic antibody clones with the desiredactivity or activities. In principle, synthetic antibody clones areselected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of the antibodiesof the invention can be obtained by designing a suitable antigenscreening procedure to select for the phage clone of interest followedby construction of a antibody clone using the Fv sequences from thephage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3. An exemplary method for generating antibodies is disclosed inthe Examples.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g., asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g., as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of clones targeting a particular antigen is desired, theindividual is immunized with antigen to generate an antibody response,and spleen cells and/or circulating B cells other peripheral bloodlymphocytes (PBLs) are recovered for library construction. In apreferred embodiment, a human antibody gene fragment library biased infavor of antigen-reactive clones is obtained by generating an antibodyresponse in transgenic mice carrying a functional human immunoglobulingene array (and lacking a functional endogenous antibody productionsystem) such that antigen immunization gives rise to B cells producinghuman antibodies against antigen. The generation of humanantibody-producing transgenic mice is described below.

Additional enrichment for antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation with antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the individual to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128:119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21:2265-2266 (1993). The in vivo recombinationapproach exploits the two-chain nature of Fab fragments to overcome thelimit on library size imposed by E. coli transformation efficiency.Naive VH and VL repertoires are cloned separately, one into a phagemidand the other into a phage vector. The two libraries are then combinedby phage infection of phagemid-containing bacteria so that each cellcontains a different combination and the library size is limited only bythe number of cells present (about 10¹² clones). Both vectors contain invivo recombination signals so that the VH and VL genes are recombinedonto a single replicon and are co-packaged into phage virions. Thesehuge libraries provide large numbers of diverse antibodies of goodaffinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88:7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20:3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (k_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutations can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1:11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 96/07754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10:779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

Nucleic acid sequence encoding the desired target antigen can bedesigned using the amino acid sequence of the desired region of antigen.

Nucleic acids encoding target antigen can be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,chemical synthesis by any of the methods described in Engels et al.,Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as the triester,phosphite, phosphoramidite and H-phosphonate methods. In one embodiment,codons preferred by the expression host cell are used in the design ofthe antigen encoding DNA. Alternatively, DNA encoding the antigen can beisolated from a genomic or cDNA library.

Following construction of the DNA molecule encoding the antigen, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are furtherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding the antigen is operably linked to asecretory leader sequence resulting in secretion of the expressionproduct by the host cell into the culture medium. Examples of secretoryleader sequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce the antigen can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce the antigen can be cultured ina variety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of antigen may be accomplished using art-recognizedmethods, some of which are described herein.

The purified antigen can be attached to a suitable matrix such asagarose beads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the antigen protein to the matrix can be accomplished bythe methods described in Methods in Enzymology, vol. 44 (1976). Acommonly employed technique for attaching protein ligands topolysaccharide matrices, e.g. agarose, dextran or cellulose, involvesactivation of the carrier with cyanogen halides and subsequent couplingof the peptide ligand's primary aliphatic or aromatic amines to theactivated matrix.

Alternatively, antigen can be used to coat the wells of adsorptionplates, expressed on host cells affixed to adsorption plates or used incell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other art-known method forpanning phage display libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigen antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting antigen, rare high affinity phage couldbe competed out. To retain all the higher affinity mutants, phages canbe incubated with excess biotinylated antigen, but with the biotinylatedantigen at a concentration of lower molarity than the target molaraffinity constant for antigen. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

Antigen clones may be activity selected. Fv clones corresponding to suchantigen antibodies can be selected by (1) isolating antigen clones froma phage library as described above, and optionally amplifying theisolated population of phage clones by growing up the population in asuitable bacterial host; (2) selecting antigen and a second proteinagainst which blocking and non-blocking activity, respectively, isdesired; (3) adsorbing the antigen binding phage clones to immobilizedantigen; (4) using an excess of the second protein to elute anyundesired clones that recognize antigen-binding determinants whichoverlap or are shared with the binding determinants of the secondprotein; and (5) eluting the clones which remain adsorbed following step(4). Optionally, clones with the desired blocking/non-blockingproperties can be further enriched by repeating the selection proceduresdescribed herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones of the invention is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide primersdesigned to specifically amplify the heavy and light chain codingregions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130:151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g., the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding antibody derived from a hybridoma of the invention can alsobe modified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g., as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). DNAencoding a hybridoma or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies of the invention.

Antigen Specificity

The present invention is applicable to antibodies of any appropriateantigen binding specificity. Preferably, the antibodies of the inventionare specific to antigens that are biologically important polypeptides.More preferably, the antibodies of the invention are useful for therapyor diagnosis of diseases or disorders in a mammal. Non-limiting examplesof therapeutic antibodies include anti-VEGF, anti-c-met, anti-IgE,anti-CD11, anti-CD18, anti-CD40, anti-tissue factor (TF), anti-HER2, andanti-TrkC antibodies. Antibodies directed against non-polypeptideantigens (such as tumor-associated glycolipid antigens) are alsocontemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor, such as a receptor tyrosine kinase) or a ligand such asa growth factor. Exemplary antigens include molecules such as renin; agrowth hormone, including human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factors such as factor VIIIC,factor IX, tissue factor (TF), and von Willebrands factor; anti-clottingfactors such as Protein C; atrial natriuretic factor; lung surfactant; aplasminogen activator, such as urokinase or human urine or tissue-typeplasminogen activator (t-PA); bombesin; thrombin; hemopoietic growthfactor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); a serum albumin such ashuman serum albumin; Muellerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; DNase; IgE; a cytotoxicT-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;activin; vascular endothelial growth factor (VEGF); receptors forhormones or growth factors; protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-β; platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19, CD20 and CD40; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

Exemplary antigens for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD46;members of the ErbB receptor family such as the EGF receptor, HER2, HER3or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95,VLA-4, ICAM-1, VCAM, α4/β7 integrin, and αv/β3 integrin including eitherα or β subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; tissue factor (TF); TGF-βalpha interferon (α-IFN); an interleukin, such as IL-8; IgE; blood groupantigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA-4; protein C etc. In some embodiments, theantibody of the invention binds (in some embodiments, specificallybinds) c-met.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos.5,571,894 and 5,587,458). Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may also be a“linear antibody,” e.g., as described, for example, in U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific orbispecific.

Accordingly, in some embodiment, the anti-c-met antibody is a one-armedantibody (i.e., the heavy chain variable domain and the light chainvariable domain form a single antigen binding arm) comprising an Fcregion, wherein the Fc region comprises a first and a second Fcpolypeptide, wherein the first and second Fc polypeptides are present ina complex and form a Fc region that increases stability of said antibodyfragment compared to a Fab molecule comprising said antigen binding arm.One armed antibodies are further described herein.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequences(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g., rodent, antibodies, where the human antibody hassimilar affinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting,”either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forantigen and the other is for any other antigen. Exemplary bispecificantibodies may bind to two different epitopes of the antigen. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress antigen. These antibodies possess a antigen-binding arm and anarm which binds the cytotoxic agent (e.g., saporin, anti-interferon-α,vinca alkaloid, ricin A chain, methotrexate or radioactive isotopehapten). Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g., F(ab′)₂bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g., tyrosine or tryptophan) (knobs orprotuberances). Compensatory “cavities” (holes) of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. Knobs and holes are further described herein.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

In one aspect, the invention provides an antibody fragment comprising atleast one characteristic that promotes heterodimerization, whileminimizing homodimerization, of the Fc sequences within the antibodyfragment. Such characteristic(s) improves yield and/or purity and/orhomogeneity of the immunoglobulin populations obtainable by methods ofthe invention as described herein. In one embodiment, a first Fcpolypeptide and a second Fc polypeptide meet/interact at an interface.In some embodiments wherein the first and second Fc polypeptides meet atan interface, the interface of the second Fc polypeptide (sequence)comprises a protuberance (also termed a “knob”) which is positionable ina cavity (also termed a “hole”) in the interface of the first Fcpolypeptide (sequence). In one embodiment, the first Fc polypeptide hasbeen altered from a template/original polypeptide to encode the cavityor the second Fc polypeptide has been altered from a template/originalpolypeptide to encode the protuberance, or both. In one embodiment, thefirst Fc polypeptide has been altered from a template/originalpolypeptide to encode the cavity and the second Fc polypeptide has beenaltered from a template/original polypeptide to encode the protuberance.In one embodiment, the interface of the second Fc polypeptide comprisesa protuberance which is positionable in a cavity in the interface of thefirst Fc polypeptide, wherein the cavity or protuberance, or both, havebeen introduced into the interface of the first and second Fcpolypeptides, respectively. In some embodiments wherein the first andsecond Fc polypeptides meet at an interface, the interface of the firstFc polypeptide (sequence) comprises a protuberance which is positionablein a cavity in the interface of the second Fc polypeptide (sequence). Inone embodiment, the second Fc polypeptide has been altered from atemplate/original polypeptide to encode the cavity or the first Fcpolypeptide has been altered from a template/original polypeptide toencode the protuberance, or both. In one embodiment, the second Fcpolypeptide has been altered from a template/original polypeptide toencode the cavity and the first Fc polypeptide has been altered from atemplate/original polypeptide to encode the protuberance. In oneembodiment, the interface of the first Fc polypeptide comprises aprotuberance which is positionable in a cavity in the interface of thesecond Fc polypeptide, wherein the protuberance or cavity, or both, havebeen introduced into the interface of the first and second Fcpolypeptides, respectively.

In one embodiment, the protuberance and cavity each comprise a naturallyoccurring amino acid residue. In one embodiment, the Fc polypeptidecomprising the protuberance is generated by replacing an originalresidue from the interface of a template/original polypeptide with animport residue having a larger side chain volume than the originalresidue. In one embodiment, the Fc polypeptide comprising theprotuberance is generated by a method comprising a step whereinpolynucleotide encoding an original residue from the interface of saidpolypeptide is replaced with polynucleotide encoding an import residuehaving a larger side chain volume than the original. In one embodiment,the original residue is threonine. In one embodiment, the originalresidue is T366. In one embodiment, the import residue is arginine (R).In one embodiment, the import residue is phenylalanine (F). In oneembodiment, the import residue is tyrosine (Y). In one embodiment, theimport residue is tryptophan (W). In one embodiment, the import residueis R, F, Y or W. In one embodiment, a protuberance is generated byreplacing two or more residues in a template/original polypeptide. Inone embodiment, the Fc polypeptide comprising a protuberance comprisesreplacement of threonine at position 366 with tryptophan, amino acidnumbering according to the EU numbering scheme of Kabat et al. (pp.688-696 in Sequences of proteins of immunological interest, 5th ed.,Vol. 1 (1991; NIH, Bethesda, Md.)).

In some embodiments, the Fc polypeptide comprising a cavity is generatedby replacing an original residue in the interface of a template/originalpolypeptide with an import residue having a smaller side chain volumethan the original residue. For example, the Fc polypeptide comprisingthe cavity may be generated by a method comprising a step whereinpolynucleotide encoding an original residue from the interface of saidpolypeptide is replaced with polynucleotide encoding an import residuehaving a smaller side chain volume than the original. In one embodiment,the original residue is threonine. In one embodiment, the originalresidue is leucine. In one embodiment, the original residue is tyrosine.In one embodiment, the import residue is not cysteine (C). In oneembodiment, the import residue is alanine (A). In one embodiment, theimport residue is serine (S). In one embodiment, the import residue isthreonine (T). In one embodiment, the import residue is valine (V). Acavity can be generated by replacing one or more original residues of atemplate/original polypeptide. For example, in one embodiment, the Fcpolypeptide comprising a cavity comprises replacement of two or moreoriginal amino acids selected from the group consisting of threonine,leucine and tyrosine. In one embodiment, the Fc polypeptide comprising acavity comprises two or more import residues selected from the groupconsisting of alanine, serine, threonine and valine. In someembodiments, the Fc polypeptide comprising a cavity comprisesreplacement of two or more original amino acids selected from the groupconsisting of threonine, leucine and tyrosine, and wherein said originalamino acids are replaced with import residues selected from the groupconsisting of alanine, serine, threonine and valine. In someembodiments, an original amino acid that is replaced is T366, L368and/or Y407. In one embodiment, the Fc polypeptide comprising a cavitycomprises replacement of threonine at position 366 with serine, aminoacid numbering according to the EU numbering scheme of Kabat et al.supra. In one embodiment, the Fc polypeptide comprising a cavitycomprises replacement of leucine at position 368 with alanine, aminoacid numbering according to the EU numbering scheme of Kabat et al.supra. In one embodiment, the Fc polypeptide comprising a cavitycomprises replacement of tyrosine at position 407 with valine, aminoacid numbering according to the EU numbering scheme of Kabat et al.supra. In one embodiment, the Fc polypeptide comprising a cavitycomprises two or more amino acid replacements selected from the groupconsisting of T366S, L368A and Y407V, amino acid numbering according tothe EU numbering scheme of Kabat et al. supra. In some embodiments ofthese antibody fragments, the Fc polypeptide comprising the protuberancecomprises replacement of threonine at position 366 with tryptophan,amino acid numbering according to the EU numbering scheme of Kabat etal. supra.

In one embodiment, the antibody comprises Fc mutations constituting“knobs” and “holes” as described in WO2005/063816. For example, a holemutation can be one or more of T366A, L368A and/or Y407V in an Fcpolypeptide, and a knob mutation can be T366W.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid (at least two, at least three, atleast 4 or more) residue in the antibody molecule replaced by adifferent residue. The sites of greatest interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table A underthe heading of “preferred substitutions.” If such substitutions resultin a change in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table A, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE A Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trn, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way infor generating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.,6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g., asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, e.g., in the Fc region. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351concerning other examples of Fc region variants. WO00/42072 (Presta) andWO 2004/056312 (Lowman) describe antibody variants with improved ordiminished binding to FcRs. The content of these patent publications arespecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased halflives and improved binding to the neonatal Fc receptor (FcRn), which isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)), are described in US2005/0014934A1 (Hinton et al.). Theseantibodies comprise an Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. Polypeptide variantswith altered Fc region amino acid sequences and increased or decreasedClq binding capability are described in U.S. Pat. No. 6,194,551B1,WO99/51642. The contents of those patent publications are specificallyincorporated herein by reference. See, also, Idusogie et al., J.Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Screening for Antibodies with Desired Properties

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art (some of which are disclosed herein). For example, theantibodies can be further characterized by a series of assays including,but not limited to, N-terminal sequencing, amino acid analysis,non-denaturing size exclusion high pressure liquid chromatography(HPLC), mass spectrometry, ion exchange chromatography and papaindigestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative assays are provided below in theExamples section.

Vectors, Host Cells, and Recombinant Methods

For recombinant production of a heterologous polypeptide (e.g, anantibody), the nucleic acid encoding it is isolated and inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. DNA encoding the polypeptide (eg, antibody) is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The choice of vector depends in part on the host cell to beused. Generally, preferred host cells are of either prokaryotic origin.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

A. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of thepolypeptide (e.g., antibody) of the invention can be obtained usingstandard recombinant techniques. Desired polynucleotide sequences may beisolated and sequenced from antibody producing cells such as hybridomacells. Alternatively, polynucleotides can be synthesized usingnucleotide synthesizer or PCR techniques. Once obtained, sequencesencoding the polypeptides are inserted into a recombinant vector capableof replicating and expressing heterologous polynucleotides inprokaryotic hosts. Many vectors that are available and known in the artcan be used for the purpose of the present invention. Selection of anappropriate vector will depend mainly on the size of the nucleic acidsto be inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various components,depending on its function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides. The vector components generally include, butare not limited to: an origin of replication, a selection marker gene, apromoter, a ribosome binding site (RBS), a signal sequence, theheterologous nucleic acid insert and a transcription terminationsequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-lactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al., (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the signal sequences native to theheterologous polypeptides, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the signalpolypeptides of the present invention. In addition, the vector maycomprise a signal sequence selected from the group consisting of thealkaline phosphatase, penicillinase, Lpp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA, and MBP.

In one aspect of the invention, one or more polynucleotides (e.g.,expression vectors) collectively encode a one-armed antibody. In oneembodiment, a single polynucleotide encodes (a) the light and heavychain components of the one armed antibody, and (b) the Fc polypeptide.In one embodiment, a single polynucleotide encodes the light and heavychain components of the one armed antibody, and a separatepolynucleotide encodes the Fc polypeptide. In one embodiment, separatepolynucleotides encode the light chain component of the one-armedantibody, the heavy chain component of the one-armed antibody and the Fcpolypeptide, respectively. Production of a one-armed antibody isdescribed in, for example, in WO2005063816.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635) and strains 63C1 and 64B4. Other strainsand derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B,E. coliλ, 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608) are alsosuitable. These examples are illustrative rather than limiting. Methodsfor constructing derivatives of any of the above-mentioned bacteriahaving defined genotypes are known in the art and described in, forexample, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147) or media described inWO2002/061090. A variety of other inducers may be used, according to thevector construct employed, as is known in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.,(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun,(2000) J. Biol. Chem. 275:17106-17113; Arie et al., (2001) Mol.Microbiol. 39:199-210. In some embodiments, DsbA and C are expressed inthe bacterial host cell.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI, and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al.,(1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou etal., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance,2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal., (1983) J. Immunol. Meth. 62:1-13. The solid phase to which ProteinA is immobilized is preferably a column comprising a glass or silicasurface, more preferably a controlled pore glass column or a silicicacid column. In some applications, the column has been coated with areagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the antibodiesdescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al., (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al., (1998)Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342).The toxins may effect their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition. Some cytotoxic drugs tend to be inactive or less active whenconjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al., (2002) Blood 99(12):4336-42; Witzig etal., (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al., (2002) J.Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity againstB-cell non-Hodgkin's Lymphoma (NHL), administration results in severeand prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumabozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composedof a hu CD33 antibody linked to calicheamicin, was approved in 2000 forthe treatment of acute myeloid leukemia by injection (Drugs of theFuture (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089;5,606,040; 5,6937,62; 5,739,116; 5,767,285; 5,773,001). Cantuzumabmertansine (Immunogen, Inc.), an antibody drug conjugate composed of thehuC242 antibody linked via the disulfide linker SPP to the maytansinoiddrug moiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al., (2003) Nature Biotechnology21(7):778-784) and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (e.g., above). Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides,”volume 1, pp. 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483 and5,780,588; Pettit et al., (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al., (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., etal., Synthesis, 1996, 719-725; and Pettit et al., (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat. Biotechnol.21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands,” U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394 and 5,770,710, as well as esperamicins (U.S.Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, catantigen B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibodyby introducing one, two, three, four, or more cysteine residues (e.g.,preparing mutant antibodies comprising one or more non-native cysteineamino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g., withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g., by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to theindividual, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g., avidin) which is conjugated to a cytotoxic agent (e.g., aradionucleotide).

Pharmaceutical Formulations

Therapeutic formulations of the heterologous polypeptide are preparedfor storage by mixing the heterologous polypeptide having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,histidine and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

A heterologous polypeptide of the present invention may be used, forexample, to purify, detect, and target a specific polypeptide itrecognizes, including both in vitro and in vivo diagnostic andtherapeutic methods.

In one aspect, an antibody of the invention can be used in immunoassaysfor qualitatively and quantitatively measuring specific antigens inbiological samples. Conventional methods for detecting antigen-antibodybinding includes, for example, an enzyme linked immunosorbent assay(ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. Manymethods may use a label bound to the antibody for detection purposes.The label used with the antibody is any detectable functionality thatdoes not interfere with its binding to antibody. Numerous labels areknown, including the radioisotopes ³²P, ³²S, ¹⁴C, ¹²⁵, I ³H and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and xanthineoxidase, lactoperoxidase, biotin/avidin, spin labels, bacteriophagelabels, stable free radicals, imaging radionuclides (such as Technecium)and the like.

Conventional methods are available to bind these labels covalently tothe heterologous polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al. Nature 144: 945 (1962); David et al.Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods40:219-230 (1981); and Nygren Histochem. and Cytochem 30:407-412 (1982).Preferred labels herein are enzymes such as horseradish peroxidase andalkaline phosphatase. The conjugation of such label, including theenzymes, to the antibody polypeptide is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166. Such bonding methods aresuitable for use with the heterologous polypeptides of this invention.

Alternative to labeling the heterologous polypeptide, antigen can beassayed in biological fluids by a competition immunoassay utilizing acompeting antigen standard labeled with a detectable substance and anunlabeled heterologous polypeptide. In this assay, the biologicalsample, the labeled antigen standards and the heterologous polypeptideare combined and the amount of labeled antigen standard bound to theunlabeled heterologous polypeptide is determined. The amount of testedantigen in the biological sample is inversely proportional to the amountof labeled antigen standard bound to the heterologous polypeptide.

In one aspect, a heterologous polypeptide (such as an antibody) of theinvention is particularly useful to detect and profile expressions ofspecific surface antigens in vitro or in vivo. As discussed before,generally, an aglycosylated antibody does not exert effector functions(i.e., ADCC or CDC activity). Therefore, when the antibody binds to thecell surface antigen, it will not initiate undesirable cytotoxic events.The surface antigen can be specific to a particular cell or tissue type,therefore serving as a marker of the cell or tissue type. Preferably,the surface antigen marker is differentially expressed at variousdifferentiation stages of particular cell or tissue types. The antibodydirected against such surface antigen can thus be used for the screeningof cell or tissue populations expressing the marker. For example, theantibody of the invention can be used for the screening and isolation ofstem cells such as embryonic stem cells, hematopoietic stem cells andmesenchymal stem cells. The antibody of the invention can also be usedto detect tumor cells expressing tumor-associated surface antigens suchc-met, HER2, HER3 or HER4 receptors.

An antibody or other heterologous polypeptide of the invention may beused as an affinity purification agent. In this process, the polypeptideis immobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized polypeptide iscontacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide. Finally,the support is washed with another suitable solvent, such as glycinebuffer, pH 5.0, that will release the antigen from the polypeptide.

In one aspect, the invention provides uses of a heterologous polypeptidegenerated using the methods of the invention, in the preparation of amedicament for the therapeutic and/or prophylactic treatment of adisease, such as a cancer, a tumor, a cell proliferative disorder,and/or an immune (such as autoimmune) disorder. The heterologouspolypeptide can be of any form described herein, including antibody,antibody fragment, polypeptide (e.g., an oligopeptide), or combinationthereof. In some embodiments, the antigen is a human protein moleculeand the subject is a human subject.

The heterologous polypeptides of the invention can be used to diagnose,treat, inhibit or prevent diseases, disorders or conditions associatedwith abnormal expression and or activity of one or more antigenmolecules, including but not limited to malignant and benign tumors;non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

In certain embodiments, an immunoconjugate comprising the antibody isadministered to the subject. Preferably, the immunoconjugate and/orantigen to which it is bound is/are internalized by the cell.

Heterologous polypeptides of the present invention can be used eitheralone or in combination with other compositions in a therapy. Forinstance, the heterologous polypeptide may be co-administered with anantibody, chemotherapeutic agent(s) (including cocktails ofchemotherapeutic agents), other cytotoxic agent(s), anti-angiogenicagent(s), cytokines, and/or growth inhibitory agent(s). Where theheterologous polypeptide inhibits tumor growth, it may be particularlydesirable to combine the heterologous polypeptide with one or more othertherapeutic agent(s) which also inhibits tumor growth. Alternatively, oradditionally, the patient may receive combined radiation therapy (e.g.external beam irradiation or therapy with a radioactive labeled agent,such as an antibody). Such combined therapies noted above includecombined administration (where the two or more agents are included inthe same or separate formulations), and separate administration, inwhich case, administration of the antibody can occur prior to, and/orfollowing, administration of the adjunct therapy or therapies.

The heterologous polypeptide (and optionally, an adjunct therapeuticagent) is/are administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibody is suitably administered by pulse infusion, particularly withdeclining doses of the antibody. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

The heterologous polypeptide composition of the invention will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of antibody present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody (when used alone or in combination with other agents suchas chemotherapeutic agents) will depend on the type of disease to betreated, the type of antibody, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments. Depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. The preferred dosage of theantibody will be in the range from about 0.05 mg/kg to about 10 mg/kg.An initial higher loading dose, followed by one or more lower doses maybe administered. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is a antibody of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice, such as cancer. Moreover, the articleof manufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises a antibody; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat cancer. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

EXAMPLES

Materials and Methods

Bacterial strains and media—The strains and plasmids used in this studyare listed in Table 1. For shake flask cultures, all strains were grownin Lauria-Bertani (LB) or C.R.A.P. phosphate-limiting media (1) at 30 or37° C. where indicated. Fermentor medium was essentially as described inreference 1. Antibiotics were added at the following concentrations: 50μg/mL carbenicillin, 50 μg/mL kanamycin, 12.5 μg/mL chloramphenicol, or20 μg/mL tetracycline.

Construction and evaluation of relative TIR libraries—The heat-stableenterotoxin II (stII), maltose-binding periplasmic protein (malE),alkaline phosphatase (phoA), or thiol:disulfide interchange protein(dsbA) signal peptides were PCR-amplified and fused to the mature domainof the phoA gene using degenerate primers that introduced wobble-basedsilent codon mutations (2) in the first six amino acids after theparental gene's initiation codon with a BssHII, MluI, or XbaIrestriction site nine base pairs (bps) upstream of said initiation codon(see Table 2). DNAs encoding for the wild-type codons of each signalsequence were also generated. These inserts were then routinely clonedinto the SpelI/NotI (New England Biolabs) sites of the pPho41 (3)plasmid and transformed into competent JM109 cells (Promega), recoveredfor one hour and subcultured in 200 mL of LB supplemented withcabenicillin at 37° C. for sixteen hours and subsequently maxi-prepped(Qiagen). An aliquot of recovered cells from each library was plated onselective LB-agar plates in order to determine library size; alllibraries produced between ˜10-100× coverage over theoretical librarysizes. Purified DNA was then transformed into competent 27C7 cells andplated on LB-agar plates supplemented with carbenicillin and 100 μg/mL5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma) and grown at 37° C.for sixteen hours. Colonies that appeared light blue putativelyindicated they harbored a TIR variant displaying at least a low level ofPhoA activity, while dark blue colonies were indicative of cellsharboring strong TIR variants (4), and white colonies implied the cellswere carrying TIR variants with little to no PhoA expression; thepercentage of blue colonies on a given agar plate for each libraryranged from ˜2-70%. DNA from individual colonies displaying varying huesof blue was miniprepped (Qiagen), sequenced by SRS Analysis (Genentech,Inc.), retransformed into competent 27C7 cells and then tested for theirbasal PhoA activities as previously described (3). Briefly, colonieswere grown in selective LB at 30° C. for sixteen hours and diluted 1:100into fresh media and grown for an additional four hours at 30° C.Cultures were then normalized based on optical density (OD₅₅₀) andresuspended in strict-AP media (3), then stored at −20° C. overnight.Cells were then thawed, partially permeabilized with toluene (Sigma)treatment (5) and aerated at 37° C. for one hour. Forty microliters ofeach culture was then added to a solution containing 1 mM disodium4-nitrophenyl phosphate hexahydrate (PNPP; Promega) in 1 M Tris-HClbuffer (pH 8.0) and incubated in darkness at room temperature for onehour. Reactions were stopped with the addition of 100 μL sodiumphosphate buffer (pH 6.5) and the absorbance at 410 nm (A₄₁₀) was readwithin 20 minutes. Relative TIR strengths were calculated by firstsubtracting from each sample's A₄₁₀ the background absorbance from aculture containing empty vector (pBR322) and then dividing by thecorrected absorbance from a culture carrying the pPho41 plasmid. Allreported TIR values are the result of at least seven replicateexperiments.

Construction of antibody expression vectors—Signal peptides wereroutinely cloned into the previously described two-cistron system (1).Heavy chain signal peptide variants were created by fusing the signalpeptide of interest via splicing overlap extension-(SOE) PCR to theheavy chain of interest and cloned into BssHII/HpaI (New EnglandBiolabs) sites. Light chain signal peptide variants were similarly madeusing SOE-PCR and cloned into MluI/PacI (New England Biolabs) orXbaI/PacI (New England Biolabs) sites as specified by the individual TIRvariant nucleotide sequence (Table 2). All construct sequences wereconfirmed by SRS Analysis (Genentech, Inc).

Small scale induction and analysis—Cells were grown in 5 mL of selectiveLB supplemented with 5 mM sodium phosphate (pH 7.0) at 30° C. for 16hours. A 500 μL aliquot of cells were then used to inoculate 25 mL ofselective C.R.A.P. phosphate-limiting media and grown for 24 hours at30° C. Where indicated, cells carrying the plasmid pJJ247 were inducedwith isopropyl β-D-thiogalactoside (IPTG) to a final concentration of1.0 mM when the cells reached an OD₆₀₀˜2.0. End point whole brothsamples were taken and diluted to an OD₆₀₀ of ˜3.0 in lysis buffer (10mM Tris pH 6.8, 5 mM EDTA, 0.2 mg/mL lysozyme (Sigma), 5 mM iodoaceticacid (Sigma)) and incubated on ice for 10 minutes. Samples weresonicated, centrifuged to remove cell debris and then analyzed usingSDS-PAGE analysis (10% Bis-Tris, Invitrogen). Whole cell lysate sampleswere normalized to equivalent optical densities reduced with 0.2 Mdithiothreitol (DTT, Sigma), and analyzed using SDS-PAGE analysis. Alllanes were loaded with equivalent volumes of samples and probed usingwith either a human anti-Fc (Southern Biotech) antibody at a 1:200,000dilution or a mouse anti-κLc (Southern Biotech) antibody at a 1:200,000dilution. All antibodies were HRP-conjugated and immunoblots werevisualized using Western Lightning-ECL (PerkinElmer) and exposing themembrane to Biomax XAR Film (Kodak). Protein samples were also analyzedvia Coomassie blue staining following standard techniques.

Large scale induction—Fermentations were performed as previouslydescribed (1). Briefly, a 500 μL aliquot of cryopreserved cells from a 5mL selective LB culture was used to inoculate 500 mL of selective LB andgrown at 30° C. for 16 hours. A 10-L fermentor was then inoculated(essentially as described in ref. 1) and cells were grown to a highdensity using a computer-based algorithm to feed a concentrated glucosesolution based on fermentation demands. Where indicated, cells carryingthe plasmid pJJ247 were induced with Where indicated, cells carrying theplasmid pJJ247 were induced with isopropyl β-D-thiogalactoside (IPTG) toa final concentration of 1.0 M when the cells reached an OD₅₅₀˜200.Whole broth and normalized OD₅₅₀ samples were taken at regular timeintervals and all fermentations were terminated after 2-3 days. Culturefitness was routinely monitored using online and offline measuredparameters. Samples were analyzed using SDS-PAGE analysis as describedabove.

HPLC analysis of samples—Samples from either small or large scaleinduction experiments were analyzed for total (insoluble and soluble)heavy or light chain concentrations through a previously developedreversed-phase HPLC analysis technique (Lisa Wong, personalcommunication). Samples were analyzed for light-chain containingantibody species by a dual-column, Protein-L reverse phase based HPLCassay (Analytical Operations, Genentech, Inc.). Antibody titers wereobtained by comparing chromatogram peak areas to those of a standardcurve generated by spiking blank samples with known amounts of moleculeof interest.

TABLE 1 Strains and plasmids used in this study Reference or Strain orplasmid Relevant genotype/phenotype source E. coli strains 27C7 ΔfhuA(ΔtonA) phoAΔE15 Δ(argF-lac)169 ptr3 (3) degP41 kan^(R) ompTΔ(nmpc-fepE)64B4 W3110 ΔfhuA ΔphoA ilvG+ Δprc spr43H1 ΔdegP Laboratory stock ΔmanAlacI^(q) ΔompT JM109 e14⁻(McrA⁻) recA1 endA1 gyrA96 thi-1 hsdR17 (r_(K)⁻ Promega m_(K) ⁺) supE44 relA1 Δ(lac-proAB) [F′ traD36 proABlacI^(q)ZΔM15] Plasmids pPho41 Cb^(r) (3) pBR322 Cb^(r), Tc^(r)Laboratory stock ph5D5 Humanized 5D5 antibody (interchangeably termedLaboratory stock’ 5D5.v2 antibody) cloned into pBR322 pJJ247 E. colidsbA and dsbC under control of the tac Laboratory stock promoter in apACYC-derived vector, Km^(r) pBR-STIIHc1.0-PhoA E. coli BssHIII-ssSTIITIRv.1 fused to Δ(1- This study 22)PhoA in pPho41 pBR-STIIHc2.41-PhoA E.coli BssHIII-ssSTII TIRv.2 fused to Δ(1- This study 22)PhoA in pPho41pBR-STIIHc3.38-PhoA E. coli BssHIII-ssSTII TIRv.3 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-STIIHc4.60-PhoA E. coli BssHIII-ssSTIITIRv.4 fused to Δ(1- This study 22)PhoA in pPho41 pBR-STIIHc5.34-PhoA E.coli BssHIII-ssSTII TIRv.5 fused to Δ(1- This study 22)PhoA in pPho41pBR-STIIHc6.52-PhoA E. coli BssHIII-ssSTII TIRv.6 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-STIIHc8.36-PhoA E. coli BssHIII-ssSTIITIRv.8 fused to Δ(1- This study 22)PhoA in pPho41 pBR-STIILc1.0-PhoA E.coli MluI-ssSTII TIRv.1 fused to Δ(1-22)PhoA This study in pPho41pBR-STIILc2.74-PhoA E. coli MluI-ssSTII TIRv.2 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-STIILc3.72-PhoA E. coli MluI-ssSTII TIRv.3 fused toΔ(1-22)PhoA This study in pPho41 pBR-DsbAHc1.48-PhoA E. coliBssHIII-ssDsbA TIRv.1 fused to Δ(1- This study 22)PhoA in pPho41pBR-DsbAHc2.WT-PhoA E. coli BssHII-ssDsbA TIRv.2 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-DsbAHc3.79-PhoA E. coli BssHII-ssDsbA TIRv.3fused to Δ(1- This study 22)PhoA in pPho41 pBR-DsbAHc7.72-PhoA E. coliBssHII-ssDsbA TIRv.7 fused to Δ(1- This study 22)PhoA in pPho41pBR-DsbALc1.WT-PhoA E. coli MluI-ssDsbA TIRv.1 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-DsbALc2.3-PhoA E. coli MluI-ssDsbA TIRv.2 fused toΔ(1-22)PhoA This study in pPho41 pBR-DsbALc3.37-PhoA E. coli MluI-ssDsbATIRv.3 fused to Δ(1-22)PhoA This study in pPho41 pBR-PhoAHc1.70-PhoA E.coli BssHII-ssPhoA TIRv.1 fused to Δ(1- This study 22)PhoA in pPho41pBR-PhoAHc2.64-PhoA E. coli BssHII-ssPhoA TIRv.2 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-PhoAHc3.WT-PhoA E. coli BssHII-ssPhoA TIRv.3fused to Δ(1- This study 22)PhoA in pPho41 pBR-PhoAHc4.67-PhoA E. coliBssHII-ssPhoA TIRv.4 fused to Δ(1- This study 22)PhoA in pPho41pBR-PhoAHc5.71-PhoA E. coli BssHII-ssPhoA TIRv.5 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-PhoAHc6.77-PhoA E. coli BssHII-ssPhoA TIRv.6fused to Δ(1- This study 22)PhoA in pPho41 pBR-PhoALc1.104-PhoA E. coliMluI-ssPhoA TIRv.1 fused to Δ(1-22)PhoA This study in pPho41pBR-PhoAXb2.41-PhoA E. coli XbaI-ssPhoA TIRv.2 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-PhoAXb3.WT-PhoA E. coli XbaI-ssPhoA TIRv.3 fused toΔ(1-22)PhoA This study in pPho41 pBR-PhoAXb5.53-PhoA E. coli XbaI-ssPhoATIRv.5 fused to Δ(1-22)PhoA This study in pPho41 pBR-PhoAXb6.15-PhoA E.coli XbaI-ssPhoA TIRv.6 fused to Δ(1-22)PhoA This study in pPho41pBR-PhoAXb7.1-PhoA E. coli XbaI-ssPhoA TIRv.7 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-PhoAXb8.24-PhoA E. coli XbaI-ssPhoA TIRv.8 fused toΔ(1-22)PhoA This study in pPho41 pBR-PhoAXb10.23-PhoA E. coliXbaI-ssPhaA TIRv.10 fused to Δ(1- This study 22)PhoA in pPho41pBR-MalEHc1.92-PhoA E. coli BssHII-ssMalE TIRv.1 fused to Δ(1- Thisstudy 22)PhoA in pPho41 pBR-MalEHc2.100-PhoA E. coli BssHII-ssMalETIRv.2 fused to Δ(1- This study 22)PhoA in pPho41 pBR-MalELc1.97-PhoA E.coli MluI-ssMalE TIRv.1 fused to Δ(1-22)PhoA This study in pPho41pBR-MalELc2.123-PhoA E. coli MluI-ssMalE TIRv.2 fused to Δ(1-22)PhoAThis study in pPho41 pBR-MalEXb1.WT-PhoA E. coli XbaI-ssMalE TIRv.1fused to Δ(1-22)PhoA This study in pPho41 pBR-MalEXb2.15-PhoA E. coliXbaI-ssMalE TIRv.2 fused to Δ(1-22)PhoA This study in pPho41pBR-MalEXb3.12-PhoA E. coli XbaI-ssMalE TIRv.3 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-MalEXb5.37-PhoA E. coli XbaI-ssMalE TIRv.5 fused toΔ(1-22)PhoA This study in pPho41 pBR-MalEXb6.4-PhoA E. coli XbaI-ssMalETIRv.6 fused to Δ(1-22)PhoA This study in pPho41 pBR-MalEXb7.25-PhoA E.coli XbaI-ssMalE TIRv.7 fused to Δ(1-22)PhoA This study in pPho41pBR-MalEXb8.13-PhoA E. coli XbaI-ssMalE TIRv.8 fused to Δ(1-22)PhoA Thisstudy in pPho41 pBR-MalEXb11.34-PhoA E. coli XbaI-ssMalE TIRv.1 1 fusedto Δ(1-22)PhoA This study in pPho41 pBR-SS-5D5-1.1 STII TIRv.1 fused to5D5 Hc, STII TIRv.1 fused to This study 5D5 Lc pBR-SS-5D5-1.2 STIITIRv.1 fused to 5D5 Hc, STII TIRv.2 fused to This study 5D5 LcpBR-SS-5D5-2.1 STII TIRv.2 fused to 5D5 Hc, STII TIRv.1 fused to Thisstudy 5D5 Lc pBR-SS-5D5-2.2 STII TIRv.2 fused to 5D5 Hc, STII TIRv.2fused to This study 5D5 Lc pBR-SM-5D5-1.1 STII TIRv.1 fused to 5D5 Hc,MalE TIRv.1 fused This study to 5D5 Lc pBR-SM-5D5-1.2 STII TIRv.1 fusedto 5D5 Hc, MalE TIRv.2 fused This study to 5D5 Lc pBR-SM-5D5-2.1 STIITIRv.2 fused to 5D5 Hc, MalE TIRv.1 fused This study to 5D5 LcpBR-SM-5D5-2.2 STII TIRv.2 fused to 5D5 Hc, MalE TIRv.2 fused This studyto 5D5 Lc pBR-SD-5D5-1.1 STII TIRv.1 fused to 5D5 Hc, DsbA TIRv.1 fusedThis study to 5D5 Lc pBR-SD-5D5-1.2 STII TIRv.1 fused to 5D5 Hc, DsbATIRv.2 fused This study to 5D5 Lc pBR-SD-5D5-2.1 STII TIRv.2 fused to5D5 Hc, DsbA TIRv.1 fused This study to 5D5 Lc pBR-SD-5D5-2.2 STIITIRv.2 fused to 5D5 Hc, DsbA TIRv.2 fused This study to 5D5 LcpBR-SP-5D5-1.1 STII TIRv.1 fused to 5D5 Hc, PhoA TIRv.1 fused This studyto 5D5 Lc pBR-SP-5D5-1.2 STII TIRv.1 fused to 5D5 Hc, PhoA TIRv.2 fusedThis study to 5D5 Lc pBR-SP-5D5-2.1 STII TIRv.2 fused to 5D5 Hc, PhoATIRv.1 fused This study to 5D5 Lc pBR-SP-5D5-2.2 STII TIRv.2 fused to5D5 Hc, PhoA TIRv.2 fused This study to 5D5 Lc pBR-MS-5D5-1.1 MalETIRv.1 fused to 5D5 Hc, STII TIRv.1 fused This study to 5D5 LcpBR-MS-5D5-1.2 MalE TIRv.1 fused to 5D5 Hc, STII TIRv.2 fused This studyto 5D5 Lc pBR-MS-5D5-2.1 MalE TIRv.2 fused to 5D5 Hc, STII TIRv.1 fusedThis study to 5D5 Lc pBR-MS-5D5-2.2 MalE TIRv.2 fused to 5D5 Hc, STIITIRv.2 fused This study to 5D5 Lc pBR-MM-5D5-1.1 MalE TIRv.1 fused to5D5 Hc, MalE TIRv.1 fused This study to 5D5 Lc pBR-MM-5D5-1.2 MalETIRv.1 fused to 5D5 Hc, MalE TIRv.2 fused This study to 5D5 LcpBR-MM-5D5-2.1 MalE TIRv.2 fused to 5D5 Hc, MalE TIRv.1 fused This studyto 5D5 Lc pBR-MM-5D5-2.2 MalE TIRv.2 fused to 5D5 Hc, MalE TIRv.2 fusedThis study to 5D5 Lc pBR-MD-5D5-1.1 MalE TIRv.1 fused to 5D5 Hc, DsbATIRv.1 fused This study to 5D5 Lc pBR-MD-5D5-1.2 MalE TIRv.1 fused to5D5 Hc, DsbA TIRv.2 fused This study to 5D5 Lc pBR-MD-5D5-2.1 MalETIRv.2 fused to 5D5 Hc, DsbA TIRv.1 fused This study to 5D5 LcpBR-MD-5D5-2.2 MalE TIRv.2 fused to 5D5 Hc, DsbA TIRv.2 fused This studyto 5D5 Lc pBR-MP-5D5-1.1 MalE TIRv.1 fused to 5D5 Hc, PhoA TIRv.1 fusedThis study to 5D5 Lc pBR-MP-5D5-1.2 MalE TIRv.1 fused to 5D5 Hc, PhoATIRv.2 fused This study to 5D5 Lc pBR-MP-5D5-2.1 MalE TIRv.2 fused to5D5 Hc, PhoA TIRv.1 fused This study to 5D5 Lc pBR-MP-5D5-2.2 MalETIRv.2 fused to 5D5 Hc, PhoA TIRv.2 fused This study to 5D5 LcpBR-DS-5D5-1.1 DsbA TIRv.1 fused to 5D5 Hc, STII TIRv.1 fused This studyto 5D5 Lc pBR-DS-5D5-1.2 DsbA TIRv.1 fused to 5D5 Hc, STII TIRv.2 fusedThis study to 5D5 Lc pBR-DS-5D5-2.1 DsbA TIRv.2 fused to 5D5 Hc, STIITIRv.1 fused This study to 5D5 Lc pBR-DS-5D5-2.2 DsbA TIRv.2 fused to5D5 Hc, STII TIRv.2 fused This study to 5D5 Lc pBR-DM-5D5-1.1 DsbATIRv.1 fused to 5D5 Hc, MalE TIRv.1 fused This study to 5D5 LcpBR-DM-5D5-1.2 DsbA TIRv.1 fused to 5D5 Hc, MalE TIRv.2 fused This studyto 5D5 Lc pBR-DM-5D5-2.1 DsbA TIRv.2 fused to 5D5 Hc, MalE TIRv.1 fusedThis study to 5D5 Lc pBR-DM-5D5-2.2 DsbA TIRv.2 fused to 5D5 Hc, MalETIRv.2 fused This study to 5D5 Lc pBR-DD-5D5-1.1 DsbA TIRv.1 fused to5D5 Hc, DsbA TIRv.1 fused This study to 5D5 Lc pBR-DD-5D5-1.2 DsbATIRv.1 fused to 5D5 Hc, DsbA TIRv.2 fused This study to 5D5 LcpBR-DD-5D5-2.1 DsbA TIRv.2 fused to 5D5 Hc, DsbA TIRv.1 fused This studyto 5D5 Lc pBR-DD-5D5-2.2 DsbA TIRv.2 fused to 5D5 Hc, DsbA TIRv.2 fusedThis study to 5D5 Lc pBR-DP-5D5-1.1 DsbA TIRv.1 fused to 5D5 Hc, PhoATIRv.1 fused This study to 5D5 Lc pBR-DP-5D5-1.2 DsbA TIRv.1 fused to5D5 Hc, PhoA TIRv.2 fused This study to 5D5 Lc pBR-DP-5D5-2.1 DsbATIRv.2 fused to 5D5 Hc, PhoA TIRv.1 fused This study to 5D5 LcpBR-DP-5D5-2.2 DsbA TIRv.2 fused to 5D5 Hc, PhoA TIRv.2 fused This studyto 5D5 Lc pBR-PS-5D5-1.1 PhoA TIRv.1 fused to 5D5 Hc, STII TIRv.1 fusedThis study to 5D5 Lc pBR-PS-5D5-1.2 PhoA TIRv.1 fused to 5D5 Hc, STIITIRv.2 fused This study to 5D5 Lc pBR-PS-5D5-2.1 PhoA TIRv.2 fused to5D5 Hc, STII TIRv.1 fused This study to 5D5 Lc pBR-PS-5D5-2.2 PhoATIRv.2 fused to 5D5 Hc, STII TIRv.2 fused This study to 5D5 LcpBR-PM-5D5-1.1 PhoA TIRv.1 fused to 5D5 Hc, MalE TIRv.1 fused This studyto 5D5 Lc pBR-PM-5D5-1.2 PhoA TIRv.1 fused to 5D5 Hc, MalE TIRv.2 fusedThis study to 5D5 Lc pBR-PM-5D5-2.1 PhoA TIRv.2 fused to 5D5 Hc, MalETIRv.1 fused This study to 5D5 Lc pBR-PM-5D5-2.2 PhoA TIRv.2 fused to5D5 Hc, MalE TIRv.2 fused This study to 5D5 Lc pBR-PD-5D5-1.1 PhoATIRv.1 fused to 5D5 Hc, DsbA TIRv.1 fused This study to 5D5 LcpBR-PD-5D5-1.2 PhoA TIRv.1 fused to 5D5 Hc, DsbA TIRv.2 fused This studyto 5D5 Lc pBR-PD-5D5-2.1 PhoA TIRv.2 fused to 5D5 Hc, DsbA TIRv.1 fusedThis study to 5D5 Lc pBR-PD-5D5-2.2 PhoA TIRv.2 fused to 5D5 Hc, DsbATIRv.2 fused This study to 5D5 Lc pBR-PP-5D5-1.1 PhoA TIRv.1 fused to5D5 Hc, PhoA TIRv.1 fused This study to 5D5 Lc pBR-PP-5D5-1.2 PhoATIRv.1 fused to 5D5 Hc, PhoA TIRv.2 fused This study to 5D5 LcpBR-PP-5D5-2.1 PhoA TIRv.2 fused to 5D5 Hc, PhoA TIRv.1 fused This studyto 5D5 Lc pBR-PP-5D5-2.2 PhoA TIRv.2 fused to 5D5 Hc, PhoA TIRv.2 fusedThis study to 5D5 Lc Hc = heavy chain Lc = light chain 5D5 = anti-c-metmonoclonal antibody clone 5D5.v2. 5D5.v2 heavy and light chain sequencesare shown in FIG. 7 and are also described in, e.g., WO2006/015371; Jinet al, Cancer Res (2008) 68: 4360.

TABLE 2 signal sequence variants Parent Clone Relative TIR gene IDRelevant genotype/phenotype strength SEQ ID NO: stII SH1.2 GCGCGCATTATG

0.99 ± 0.07 1

CTTCTTGCATCTATGTTCGTTTTTTCTAT T GCTACAAACGCTTACGCT SH2.41 GCGCGCATTATG

1.94 ± 0.05 2

CTTCTTGCATCTATGTTCGTTTTTTCTA TTGCTACAAACGCTTACGCT SH3.38 GCGCGCATTATG

2.9 ± 0.2 3

C TTCTTGCATCTATGTTCGTTTTTTCTATT GC TACAAACGCTTACGCT SH4.60 GCGCGCATTATG

4.1 ± 0.1 4

CTTCTTGCATCTATGTTCGTTTTTTCTAT T GCTACAAACGCTTACGCT SH5.34 GCGCGCATTATG

5.0 ± 0.2 5

CTTCTTGCATCTATGTTCGTTTTTTCTA TTGCTACAAACGCTTACGCT SH6.52 GCGCGCATTATG

5.9 ± 0.2 6

CTTCTTGCATCTATGTTCGTTTTTTCT ATTGCTACAAACGCTTACGCT SH8.36 GCGCGCATTATG

7.7 ± 0.1 7

CTTCTTGCATCTATGTTCGTTTTTTCT ATTGCTACAAACGCTTACGCT SL1.2 ACGCGTATTATG

0.75 ± 0.07 8

CTTCTTGCATCTATGTTCGTTTTTTCT ATTGCTACAAACGCTTACGCT SL2.74 ACGCGTATTATG

1.9 ± 0.2 9

CTTCTTGCATCTATGTTCGTTTTTTCT ATTGCTACAAACGCTTACGCT SL3.72 ACGCGTATTATG

2.9 ± 0.2 10

CTTCTTGCATCTATGTTCGTTTTTTCT ATTGCTACAAACGCTTACGCT malE MH1.92GCGCGCATTATG

1.1 ± 0.1 11

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MH2.100GCGCGCATTATG

1.9 ± 0.1 12

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC ML1.97ACGCGTATTATG

1.1 ± 0.1 13

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC ML2.123ACGCGTATTATG

2.0 ± 0.1 14

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX1.wtTCTAGAATTATG

1.1 ± 0.1 15

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX2.15TCTAGAATTATG

2.0 ± 0.1 16

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX3.1TCTAGAATTATG

3.01 ± 0.09 17

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX5.37TCTAGAATTATG

5.0 ± 0.2 18

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX6.4TCTAGAATTATG

5.8 ± 0.3 19

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX7.25TCTAGAATTATG

7.1 ± 0.2 20

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX8.13TCTAGAATTATG

8.2 ± 0.3 21

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC MX11.34TCTAGAATTATG

10.8 ± 0.5  22

CGCATCCTCGCATTATCCGCATTAAC GACGATGATGTTTTCCGCCTCGGCTCTC GCC phoA PH1.70GCGCGCATTATG

1.14 ± 0.05 23

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PH2.64 GCGCGCATTATG

1.93 ± 0.03 24

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PH3.wt GCGCGCATTATG

2.8 ± 0.1 25

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PH4.67 GCGCGCATTATG

3.7 ± 0.1 26

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PH5.71 GCGCGCATTATG

5.1 ± 0.3 27

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PH6.77 GCGCGCATTATG

6.0 ± 0.4 28

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PL1.104 ACGCGTATTATG

1.00 ± 0.07 29

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PX2.41 TCTAGAATTATG

2.0 ± 0.1 30

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PX3.wt TCTAGAATTATG

3.39 ± 0.09 31

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PX5.53 TCTAGAATTATG

4.9 ± 0.1 32

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC PX6.15 TCTAGAATTATG

5.9 ± 0.2 33

CTGGCACTCTTACCGTTACTGTTTACC CCTGTGACAAAAGCC PX8.24 TCTAGAATTATG

8.0 ± 0.1 34

CTGGCACTCTTACCGTTACTGTTTACC CCTGTGACAAAAGCC PX10.23 TCTAGAATTATG

10.0 ± 0.4  35

CTGGCACTCTTACCGTTACTGTTTAC CCCTGTGACAAAAGCC dsbA DH1.48 GCGCGCATTATG

0.80 ± 0.03 36

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DH2.wt GCGCGCATTATG

1.89 ± 0.09 37

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DH3.79 GCGCGCATTATG

2.92 ± 0.08 38

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DH7.72 GCGCGCATTATG

6.7 ± 0.2 39

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DL1.wt ACGCGTATTATG

1.0 ± 0.1 40

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DL2.3 ACGCGTATTATG

1.87 ± 0.09 41

CTGGCTGGTTTAGTTTTAGCGTTTAG CGCATCGGCG DL3.37 ACGCGTATTATGAAGAAGATTTGGTTA2.6 ± 0.1 42 GCACTGGCTGGTTTAGTTTTAGCGTTTA GCGCATCGGCG Bold italics =sequence that was varied (i.e., the first six amino acids after theinitiation codon) Italic = BssHII, MluI, or XbaI restriction siteLegend: Clone naming convention is as follows: XY.# = X designates thesignal sequence (S = STII, P = PhoA and so on); Y designates therestriction sequence (H means the Bssh11 restriction site, X designatesthe XbaI site, L designates the MluI restriction site) and # designatesthe TIR strength (eg, 1 = TIR of 1, 7.72 = TIR of 7.72). wt = wildtypeTIR sequence.

TABLE 3 Final time point fermentation titers Heavy chain signal sequence(TIR)/light chain signal Relative full-length sequence (TIR) DsbA/C(+/−) Ab titer* STII (1)/STII (1) − 1.0 STII (1)/STII (1) + 4.9 STII(1)/PhoA (1) − 0.6 STII (1)/PhoA (1) + 5.6 STII (2)/STII (2) + 0.8 MalE(1)/STII (1) − 0.4 MalE (1)/PhoA (1) − 0.4 MalE (1)/PhoA (1) + 1.5 DsbA(1)/STII (1) − 1.4 DsbA (1)/STII (1) + 3.3 DsbA (1)/STII (2) + 3.6 DsbA(2)/STII (1) − 0.9 DsbA (1)/MalE (1) − 1.7 DsbA (1)/MalE (1) + 10.1 DsbA(1)/DsbA (1) − 1.9 DsbA (1)/DsbA (1) + 12.7 DsbA (2)/DsbA (2) + 10.6DsbA (l)/PhoA (1) − 1.9 DsbA (l)/PhoA (1) + 10.0 DsbA (2)/PhoA (1) − 1.5DsbA (2)/PhoA (1) + 6.7 PhoA (1)/STII (1) − 0.3 *All samples normalizedto the titer of the STII (1)/STII (1) sample, which comprised fulllength antibody expressed without chaperones DsbA and DsbC present.Results/Discussion

We developed novel variant translational initiation region (TIR) signalpeptide libraries (FIG. 2, Table 2) for signal peptides representing twoof the major secretion pathways for transport across the inner-membranein E. coli: sec (PhoA, MalE) and SRP (DsbA, STII). Each librarycomprises a panel of vectors with comprising variant TIRs of differingtranslational strengths, providing a means by which to readily adjustlevel of translation for a given protein of interest. Themaltose-binding periplasmic protein (MalE) and alkaline phosphatase(PhoA) signal peptides direct translocation from the cytoplasm to theperiplasm in a post-translational manner with the aid of the molecularmotor SecA. The heat-stable enterotoxin II (stII) and thiol:disulfideinterchange protein (dsbA) signal peptides direct translocation in aco-translational manner with aid from the signal recognition particle(SRP) (FIG. 1).

During construction of the library, a BssHII, MluI, or XbaI restrictionsite was inserted nine base pairs (bps) upstream of the parental gene'sinitiation codon. Depending upon the type of restriction site present,different ranges of TIR strengths were observed (FIG. 2). In general,sequences bearing an MluI site displayed the smallest range of TIRstrengths (˜1-3), while a BssHII site upstream allowed for a moderaterange of TIR strengths (˜1-8), and an XbaI site the highest range(˜1-11). These restriction sites present in the untranslated region areencompassed in the TIR (3). While it cannot be ruled out that higher TIRvariants may exist for any of the signal peptide/restriction sitecombinations examined, these results appear to be representative of themean TIR strengths of each signal peptide library examined.

A series of plasmids was constructed to illustrate the effect oftranslational level and signal peptide on secretion. In each case, thegene of interest was inserted downstream of the phoA promoter, trpShine-Dalgarno and a signal sequence possessing a different relative TIRstrength. Following transformation and induction of the phoA promoter atthe shake flask scale, lysates from whole cells expressing theheterologous protein, the anti-c-met antibody clone 5D5.v2, wereanalyzed by SDS-PAGE. In these experiments, either heavy chain or lightchain TIR was varied, with the corresponding light chain or heavy chain,respectively, kept invariant.

FIG. 3 shows the results of heavy chain signal peptide manipulation.When probed with an α-Fc specific antibody, the ssDsbA-heavy chain TIRone variant gave a clear increase in full-length antibody (FL-Ab), aswell as heavy-light (HL) dimer and heavy-heavy-light (HHL) species, overthe other signal peptide variants (FIG. 3A, top blot). An examination ofthe total heavy chain from these samples revealed relatively similarlevels between all signal peptide fusions examined (FIG. 3A, bottomblot). When light chain was visualized with an α-κLc antibody, similarresults were obtained, with the ssDsbA-heavy chain TIR one variant againdisplaying the highest level of FL-Ab (FIG. 3B, top blot). Strikingly,the DsbA TIR one-heavy chain fusion sample lacked the lower massspecies—the predicted light-light (LL) dimer and free light chain—seenin the other samples. Generally, in the case where thepost-translational signals (MalE, PhoA) is fused to the heavy chainthere appear to be more expressed total light chain than in the cases ofthe co-translational (STII, DsbA) signal peptide fusions (FIG. 3B,bottom blot). In general, the following hierarchy was observed withrespect to the signal peptide fused to the heavy chain and full lengthantibody production: DsbA>STII>MalE>PhoA. Notably, the DsbA variant TIRresulted in increased expression (e.g., of full length antibody)compared to STII variant TIR, even though the relative TIR strength didnot change (i.e., both TIRs were strength one).

FIG. 4 shows the results of light chain signal peptide manipulation.Changing the light chain signal peptide from an STII TIR one variant toeither a PhoA TIR one or two variant produced a noticeable increase inFL-Ab titer (FIG. 4, top blot). Modification of the signal peptide fusedto the light chain did not appear to effect the total amount of heavychain expressed (FIG. 4, middle blot), but did significantly alter thetotal amount of light chain present, with the largest accumulation ofprocessed light chain appearing in samples with a STII or DsbA TIRvariant two fused to the light chain (FIG. 4, bottom blot). When fusedto the post-translational signal peptides, two bands were observed inthe total light chain samples, indicative of unprocessed light chain. Ingeneral, the following hierarchy was observed with respect to signalpeptide fusions to the light chain and full length antibody production:PhoA≧MalE>STII>DsbA.

Monitoring assembly of antibody species over time from 10-Lfermentations revealed similar results to the shake flask experimentsshown in FIGS. 3 and 4. The highest amount of FL-Ab was observed fromsamples with a DsbA-derived TIR variant fused to the heavy chain andeither a DsbA- or PhoA-derived signal peptide fused to the light chain(FIG. 5, top blot). These samples also displayed more HHL and HL dimerspecies than did the STII TIR one heavy chain fusion. Additionally, LLdimer and free light chain was readily visible in samples with the PhoATIR one signal peptide fused to the light chain. Examination of reducedtotal protein samples revealed that the DsbA signal peptide fusionresulted in more total heavy chain than the STII fusion under theexpression inducing conditions of the fermentation (FIG. 5, middleblot). Similarly for the light chain signal peptide fusions, a higheraccumulation of light chain was observed with the DsbA TIR one signalpeptide fusions than the STII TIR one (FIG. 5, bottom blot). However,the highest accumulation of light chain was seen with the PhoA TIR onesignal peptide fusion. The two bands seen in total light chain samplestaken from the shake flasks appears as only one band in fermentationsamples, indicative of light chain being more efficiently processedduring 10-L fermentation.

We fused different signal peptides to the mature domain of the E. coliphoA gene (mPhoA) to further examine the differences in total light orheavy chain expression levels when fused to either STII- or DsbA-derivedTIR variants and expressed in shake flask cultures under inducingconditions. Similar effects on total light and heavy chain expressionlevels were observed (FIG. 6). When protein expression was induced,expression of mPhoA showed a concomitant rise with increased TIRstrength for the DsbA signal peptide fusions, up to a TIR strength ofseven (the highest TIR strength used in this study). A similar increasein mPhoA expression with TIR strength increase was observed for the STIIsignal peptide fusions up to a TIR strength of six or eight was reached,whereby the amount of mPhoA appears to decrease compared to the mPhoApresent in the STII TIR three sample. Strikingly, more heavy and lightchain was produced using a DsbA strength one TIR than using a STIIstrength one TIR, and STII-driven translocation of PhoA reached amaximum amount at a lower total protein concentration than did Dba-drivetranslocation of PhoA. Moreover, changing TIR sequence from STII to DsbAincreased the dynamic range of TIR effect.

Samples from 10-L fermentations were analyzed for antibody titers usinga Protein-L-based HPLC assay (Table 3). The HPLC data were in goodagreement with qualitative titer levels revealed by Western blotanalysis (FIGS. 3, 4). When the heavy chain signal sequence was changedfrom a STII-derived TIR variant to a Dsb-derived TIR variant, FL-Abtiters increased ˜40-90%. The highest titers were produced when a DsbAone heavy chain fusion was paired with a light chain fused to either theDsbA, MalE, or PhoA TIR one signal peptides. Highest titers weregenerated when the light chain was fused to the MalE or PhoA TIR signalpeptides.

By contrast, FL-Ab titer fell when a post-translational signal peptidewas fused to the heavy chain, with a PhoA TIR one and MalE TIR onesignal peptide fusion showing a 70% and 60% drop in titer, respectively.We concluded that heavy chain expression was optimized when aco-translational signal peptide (e.g., DsbA) was used to drivetranslation.

We tested the effect of chaperone overexpression. The overexpression ofchaperones DsbA and DsbC (sometimes termed DsbA/C herein) enhanced thebenefits of DsbA signal peptide fused to the heavy chain and DsbA, PhoA,or MalE signals fused to the light chain. When compared to expression ofFL-Ab by a STII TIR one signal fused to the heavy and light chains(SS1.1+Chaperones), an approximate 2- to 2.5-fold increase in FL-Abtiter was seen with a DsbA TIR one-heavy chain fusion coupled with aMalE, PhoA, or DsbA TIR one light chain fusion.

We examined the relationship between the signal peptide fused to thelight chain and heavy chain of an antibody and final antibody titers.Fully-assembled antibody (FL-Ab) titers were highest when aco-translational (e.g., DsbA or STII) signal peptide was fused to theN-terminus of the heavy chain, with the DsbA-derived TIR variantsresulting in the maximum observed FL-Ab yields. Thus, DsbA TIR variantsmay allow for higher translation levels of passenger protein than doSTII TIR variants under inducing conditions, thereby resulting in higherexpression levels of processed passenger protein. By contrast, antibodytiters dramatically fell when either post-translational signal peptide(i.e., MalE or PhoA) was fused to the heavy chain\. This effect may bedue to proteolysis or may be due to a different folding pathway followedby the heavy chain (6). An examination of total heavy chain levels fromsamples expressing either a PhoA or MalE TIR one signal peptide fused tothe heavy chain revealed a slight shift in apparent mass, potentiallydue to the presence of unprocessed heavy chain (FIG. 3A, bottom blot).

Fusion of post-translational signal peptide MalE-derived or PhoA-derivedTIR variants to the light chain resulted in a large accumulation ofprocessed light chain and increased antibody titers over STII-mediatedtranslocation during 10L fermentation (FIG. 5, bottom blot). Increasedyields of both light chain as well as FL-Ab were also observed when thelight chain was translocated by DsbA TIR variants as compared with lightchain translocated by STII TIR variants. However, the amount of totallight chain expressed from the DsbA TIR one variant was not a great atthat from the PhoA or MalE TIR one variants. Interestingly, analysis ofsamples taken over time from 10-L fermentations indicate that FL-Abtiters from runs with the light chain fused to either MalE, DsbA, orPhoA TIR variants continued to rise over time while fusions to STII TIRvariants reached not only a lower maximum titer, but reached that titerlevel at a much earlier time point (FIG. 5, top blot). Thus, these datasuggest that the light chain may be effectively translocated in either aco- or post-translational manner while the heavy chain requiresco-translational translocation for peak expression.

Expression of a one-armed anti-c-met antibody: We evaluated therelationship between the signal peptide fused to the light chain, heavychain and Fc of a one-armed antibody, and the final antibody titers.Plasmids were constructed using STII signal sequences with TIRs of 1 forlight chain, heavy chain, and the Fc polypeptide, using the PhoA signalsequence with a TIR of 1 (SEQ ID NO: 29) for light chain and DsbA signalsequence with a TIR of 1 (SEQ ID NO: 40) for heavy chain and the Fcfragment; and using the PhoA signal sequence with a TIR of 1 for lightchain and the Fc fragment and the DsbA signal sequence with a TIR of 1for HC. Relative titer numbers were from end of run samples and weremeasured using the Protein L-reversed phase HPLC assay described above.The relative titer values were normalized to the titer for the case inthe first row of Table 4—STII signal sequences and TIR=1 for LC, HC, andFc without the co-expression of DsbA/C.

The results are shown in Table 4. One-armed antibody relative titerswere highest when a co-translational (e.g., DsbA) signal peptide wasfused to the N-terminus of the heavy chain, post-translational (e.g.,PhoA) signal peptide was fused to the N-terminus of the light chain, anda post-translational (e.g., PhoA) signal peptide was fused to the Fcregion, and expression was in the presence of DsbA/C. In general, thefollowing hierarchy was observed with respect to the signal peptidefused to the light chain, heavy chain and Fc fragment, and one-armedantibody expression in the presence of DsbA/C: P.D.D>P.D.P.>S.S.S.Expression levels in the absence of DsbA/C were similar in all testedsamples, in which most of the antibody secreted to the periplasm wasaggregated. Co-expression of disulfide bond chaperones increased thefolded antibody produced, thus revealing the increased antibodyexpression realized by TIR optimization.

TABLE 4 Expression of monovalent one-armed anti-c-met antibody MetMAb).Relative Plasmid LC, HC, Fc DsbA/C Titer pxCM11H.v2.H.Fc.1.K.2192 STIITIR 1 for − 1.0 Lc, Hc, and Fc pxCM11H.v2.H.Fc.1.K.2192 STII TIR 1 for +1.7 Lc, Hc, and Fc pPDD.11.MetMAb PhoA TIR 1 for − 1.0 Lc, DsbA TIR 1for Hc and Fc pPDD.11.MetMAb PhoA TIR 1 for + 3.8 Lc, DsbA TIR 1 for Hcand Fc pPDP.11.MetMAb PhoA TIR 1 for − 0.7 Lc, DsbA TIR 1 for Hc, PhoATIR for Fc pPDP.11.MetMAb PhoA TIR 1 for + 2.5 Lc, DsbA TIR 1 for Hc,PhoA TIR for Fc Abbreviations: D = signal sequence DsbA P = signalsequence PhoA. XXX#.#.# (e.g. PDP.111) refers to light chain signalsequence, heavy chain signal sequence, Fc signal sequence, light chainTIR, heavy chain TIR, Fc TIR used in the experiment.

In summary, this technology offers a novel means for increasing foldedantibody yields, for example, in E. coli through manipulation of lightchain and heavy chain expression via the selection from a new array ofTIR variants and further by the use of co- or post-translational signalsequences for light chain and co-translational signal sequence for heavychain. Improved expression of one-armed antibodies comprising a heavychain, a light chain and a Fc region was also accomplished using thenovel TIR variants disclosed herein, and further by the use of co- orpost-translational signal sequences for light chain, co-translationalsignal sequence for heavy chain, and co- or post-translational signalsequence for Fc polypeptide resulted. The utility of this method appearsto be broadly applicable to a wide-range of antibodies (for example,bispecific antibodies comprising knob and hole mutations), antibodyderivatives and bacterial-based recombinant protein production as awhole.

PARTIAL REFERENCE LIST

1. Simmons, L. C., Reilly, D., Klimowski, L., Raju, T. S., Meng, G.,Sims, P., Hong, K., Shields, R. L., Damico, L. A., Rancatore, P., andYansura, D. G. (2002) Journal of immunological methods 263(1-2), 133-147

2. Stemmer, W. P., Morris, S. K., Kautzer, C. R., and Wilson, B. S.(1993) Gene 123(1), 1-7

3. Simmons, L. C., and Yansura, D. G. (1996) Nature biotechnology 14(5),629-634

4. Le Calvez, H., Green, J. M., and Baty, D. (1996) Gene 170(1), 51-55

5. Jackson, R. W., and DeMoss, J. A. (1965) Journal of bacteriology90(5), 1420-1425

6. Kadokura, H., and Beckwith, J. (2009) Cell 138(6), 1164-1173

Although the forgoing refers to particular embodiments, it will beunderstood that the present invention is not so limited. It will occurto those ordinary skilled in the art that various modifications may bemade to the disclosed embodiments without diverting from the overallconcept of the invention. All such modifications are intended to bewithin the scope of the present invention.

What is claimed is:
 1. A method of making an a heterologous polypeptide,said method comprising culturing a host cell comprising polynucleotidecomprising a variant translation initiation region (TIR) comprising asequence of one of SEQ ID NOs 1-42 operably linked to a polynucleotideencoding the heterologous polypeptide, so that the polynucleotide isexpressed, whereby upon expression of the heterologous protein in a hostcell, the heterologous polypeptide is folded to form a biologicallyactive heterologous polypeptide.
 2. The method of claim 1, wherein thevariant TIR comprises sequence of one of SEQ ID NOs. 1-14, 16-24, 26-39,41-42.
 3. A method of making an antibody, said method comprisingculturing a host cell comprising a polynucleotide comprising (1) a firstTIR operably linked to a polynucleotide encoding an antibody heavychain, wherein the TIR comprises a co-translational prokaryoticsecretion signal sequence; and (2) a second TIR operably linked to apolynucleotide encoding an antibody light chain, wherein the second TIRcomprises a co-translational or post-translational prokaryotic secretionsignal sequence, so that the antibody is expressed, whereby uponexpression of the antibody in the host cell, the heavy and light chainsare folded and assembled to form a biologically active antibody, whereinthe first and the second TIR each comprise a sequence of one of SEQ IDNOs 1-42.
 4. The method of claim 3, wherein the first translationinitiation region comprises a STII or DsbA variant signal sequence. 5.The method of claim 4, wherein the first translation initiation regioncomprises a DsbA variant signal sequence.
 6. The method of claim 4,wherein the first translation initiation region comprises sequence ofone of SEQ ID NOs: 1-10 and 36-42.
 7. The method of claim 4, wherein thefirst translation initiation region comprises sequence of one of SEQ IDNOs: 1-10 and 36-39 and 41-42.
 8. The method of claim 3, wherein thesecond translation initiation region comprises a STII, DsbA, MalE orPhoA variant signal sequence.
 9. The method of claim 8, wherein thesecond translation initiation region comprises a PhoA or MalE variantsignal sequence.
 10. The method of claim 8, wherein the secondtranslation initiation region comprises sequence of one of SEQ ID NOs.1-14, 16-24, 26-39, and 41-42.
 11. The method of claim 3, wherein thepolynucleotide encoding an antibody further comprises (3) a thirdtranslation initiation region operably linked to a polynucleotideencoding a Fc polypeptide, and wherein the third TIR comprises asequence of one of SEQ ID NOs 1-42.
 12. The method of claim 11, whereinthe third translation initiation region comprises a PhoI or DsbA variantsignal sequence.
 13. The method of claim 3, wherein the first and secondtranslational initiation regions provide approximately equaltranslational strengths.
 14. The method of claim 3, wherein thepolynucleotide further comprises a promoter.
 15. The method of claim 14,wherein the promoter is a prokaryotic promoter selected from the groupconsisting of phoA, tac, lpp, lac-lpp, lac, ara, and T7 promoter. 16.The method of claim 3, wherein the antibody is a monoclonal antibody.17. The method of claim 16, wherein the antibody is a chimeric antibody,an affinity matured antibody, a bispecific antibody, a humanizedantibody, an antibody fragment or a human antibody.
 18. The method ofclaim 17, wherein the antibody fragment is a one-armed antibody.
 19. Themethod of claim 17 or 18, wherein the antibody binds c-met.
 20. Themethod of claim 19, wherein the anti-c-met antibody comprises (a) afirst polypeptide comprising a heavy chain variable domain having thesequence:EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS(SEQ ID NO: 43), CH1 sequence, and a first Fc polypeptide; (b) a secondpolypeptide comprising a light chain variable domain having thesequence:DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR(SEQ ID NO:44), and CL1 sequence; and (c) a third polypeptide comprisinga second Fc polypeptide, wherein the heavy chain variable domain and thelight chain variable domain are present as a complex and form a singleantigen binding arm, wherein the first and second Fc polypeptides arepresent in a complex and form a Fc region that increases stability ofsaid antibody fragment compared to a Fab molecule comprising saidantigen binding arm.
 21. The method of claim 3, wherein the host cell isa prokaryotic cell.
 22. The method of claim 21, wherein the prokaryoticcell is E. coli.
 23. The method of claim 22, wherein the E. coli hostcell is a strain that is deficient in endogenous protease activities.24. The method of claim 22, wherein the E. coli host cell lacks degP andprc genes and harbors a mutant spr gene.
 25. The method of claim 3,wherein the method further comprises recovering the antibody from thehost cell culture.
 26. The method of claim 25, wherein the antibody isrecovered from the host cell culture medium.
 27. The method of claim 25or 26, further comprising combining the recovered heterologouspolypeptide with a pharmaceutically acceptable carrier, excipient, orcarrier to prepare a pharmaceutical formulation comprising theheterologous polypeptide.
 28. The method of claim 3, wherein at least50% of the immunoglobulin polypeptide complexes that are formed are theantibody.
 29. A method of secreting an antibody from a cell, said methodcomprising culturing a host cell comprising a polynucleotide comprising(1) a first TIR operably linked to a polynucleotide encoding an antibodyheavy chain, wherein the TIR comprises a co-translational prokaryoticsecretion signal sequence; and (2) a second TIR operably linked to apolynucleotide encoding an antibody light chain, wherein the second TIRcomprises a co-translational or post-translational prokaryotic secretionsignal sequence, so that the antibody is expressed and the antibody issecreted, wherein the first and the second TIR each comprise a sequenceof one of SEQ ID NOs 1-42.